Amenamevir

Profile of anti-herpetic action of ASP2151 (amenamevir) as a helicase-primase inhibitor

Abstract
The antiherpetic drugs acyclovir (ACV, valaciclovir) and penciclovir (famciclovir) are phosphorylated by viral thymidine kinase and terminate DNA synthesis. ASP2151 (amenamevir) and foscavir (PFA) directly inhibit viral helicase-primase and DNA polymerase, respectively, and inhibit replication of herpes simplex virus (HSV) and varicella-zoster virus. ACV, ASP2151, and PFA all inhibit HSV with a different mechanism of action and as a consequence, the kinetics of viral DNA accumulation and progeny virus production differ. This study focused on how viral DNA synthesis and its related events in the replication cycle would influence anti-HSV action of ACV, ASP2151, and PFA. ASP2151 suppressed HSV replication more efficiently than ACV at 10 x 50% effective concentration of plaque formation (EC50), when treatments were started 0 to 24 h after infection. ASP2151 and PFA were more potent than ACV in suppressing viral DNA synthesis and infectious virus production when they were added up to 3 hours following infection. The virus replicated in the presence of ACV was compared for the ratios of HSV DNA copy number to infectivity with that without ACV and infectivity of ACV-treated virus was less efficient than that without ACV-treatment. The EC50 of infected cells in the time course after infection was preserved in PFA, limited in ASP2151, and much increased for ACV, indicating that viral DNA synthesis had little effect on antiviral action of PFA and ASP2151 but reduced the susceptibility of ACV. ASP2151 showed a preferable profile as an anti-herpetic agent with a better pharmacokinetic profile than ACV.

1.Introduction
Herpes simplex virus (HSV) type 1 (HSV-1) and HSV-2 are widely prevalent pathogens belonging to the human herpesvirus family. Both HSVs establish life-long latent infections in sensory ganglia after the primary infection and eventually reactivate, leading to recurrent episodes. Suppressive therapy has been successfully used to reduce recurrence and viral shedding (Brown et al., 2005; Corey et al., 2004; Daikoku et al., 2016; Honda et al., 2001; Leung and Sacks, 2000; Moomaw et al., 2003; Reitano et al., 1998; Wald et al., 2006).
Synthetic nucleoside analogues targeting viral DNA synthesis, such as acyclovir (ACV), penciclovir, famciclovir, and valaciclovir, are used for treatment of HSV infections (De Clercq, 1993; De Clercq and Field, 2006). Phosphonoformic acid (PFA or foscarnet) is used for treatment of nucleoside analogue-resistant HSV infection (Balfour et al., 1994). In addition, ASP2151 (amenamevir), one of helicase-primase inhibitors (Biswas et al., 2014; Crumpacker and Schaffer, 2002; Crute et al., 2002; James et al., 2015; James and Prichard, 2014; Kleymann et al., 2002), has been reported to show antiviral activity against HSV in vitro and in vivo (Chono et al., 2010; Chono et al., 2012b; Chono et al., 2013; Himaki et al., 2012; Katsumata et al., 2013; Katsumata et al., 2011; Katsumata et al., 2012) and had good pharmacokinetic profiles for efficacy against HSV and VZV infection in human trials with a single oral daily administration (Tyring et al., 2012). ACV inhibits viral DNA synthesis by chain termination, but the efficacy depends on the ratio of deoxyguanosine-triphosphate (dGTP) and ACV-triphosphate (ACV-TP) phosphorylated by viral thymidine kinase (TK). On the other hand, PFA and ASP2151 directly inhibit DNA polymerase and helicase-primase activities, respectively. PFA competes with pyrophosphate on viral DNA polymerase, and ASP2151 inhibits helicase-primase with no competing substance. Thus, these anti-herpetic agents have similar inhibitory activities, but their modes of action are different.This study focused on how viral DNA synthesis in the replication cycle would influence anti-HSV action of ACV, ASP2151, and PFA. We compared the effects of the timing of drug treatment before and after viral DNA synthesis in the replication cycle on the inhibitory mode of viral replication by ASP2151, ACV, and PFA. Viral DNA synthesis had little effect on antiviral action of PFA and ASP2151 but reduced the susceptibility of ACV. Thus our study showed the better profile of ASP2151 by inhibiting viral replication through direct inhibition of viral helicase-primase.

2.Materials and Methods
ASP2151 was provided by Astellas Pharma Inc. (Tokyo, Japan) and Maruho Co., Ltd. ACV and PFA were purchased from Wako Pure Chemical Industries (Osaka, Japan) and Sigma-Aldrich (St. Louis, MO, USA), respectively.Human embryonic lung (HEL) cells and Vero cells were grown in a growth medium consisting of Eagle’s minimum essential medium (Nissui Biosciences, Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Chono et al., 2012b; Kurokawa et al., 2001). The medium containing 2% FBS was used for infection and propagation of the virus. HSV-1 strain 7401H was propagated using Vero cells in the maintenance medium and stored at -80°C until use (Kurokawa et al., 2001; Kurokawa et al., 1993; Sasivimolphan et al., 2009).Antiviral assay has been performed to examine the effects of viral DNA synthesis and its related cellular events on the antiviral activity of ASP2151, ACV, and PFA by changing the timing of treatment with antiviral drugs in the infected cultures. HEL cells in 24-well culture plates were infected with HSV-1 at a multiplicity of infection (MOI) of 0.4 plaque forming units (pfu)/cell and incubated at 37°C for 1 h. Infected cells were washed three times with maintenance medium [defined as 0 h post-infection (pi)] and incubated for 48 h with 0.5 ml of maintenance medium. The antiviral compound was added to the assay wells in triplicate at 0, 1, 2, 3, 4, 5, 6, 7, 8, 12, 24 h and incubated up to 48 h after infection. The concentrations of antiviral drugs were set at 10 times the 50% effective concentrations (EC50) determined by the plaque reduction assay in HEL cells. Ten times the EC50 of ASP2151, ACV, and PFA for HSV-1 were 1.00 µM, 3.31 µM, and 2.01 µM, respectively. Supernatants and cells were separately harvested at 48 h pi, and the supernatants were centrifuged to remove cell debris.

Then DNA in the supernatants and cells was extracted to determine HSV genome copy numbers by real-time quantitative PCR. Virus yields in the supernatants were determined by a plaque formation assay using Vero cells. Two hundred microliters of the culture supernatants was 10 fold serially diluted, inoculated onto Vero cells, and incubated for three days in maintenance medium containing 0.8% methyl cellulose. The cells were fixed with 5% formalin and stained with 0.03% methylene blue, and the numbers of plaques were counted. As viral DNA in the cellular fraction contains viral DNA in the virion and replicative form of viral DNA, virus titer and viral DNA in the cellular fraction would not correlate and was not determined.HEL cells were infected with HSV-1 at an MOI of 0.4 pfu/cell and incubated at 37 °C for 1 h. Infected cells were washed three times with maintenance medium, and 0.5 ml of maintenance medium containing one of the antiviral compounds ACV, ASP2151, or PFA at 10 times its EC50 value or ACV at 100 times its EC50 value was added. The supernatants andcells were separately harvested at 0, 4, 8, 16, 24, and 48 h after infection, and HSV genome copy numbers in the supernatants and cellular fraction and virus yields in the supernatants were determined as described above. The ratios of HSV DNA copy number to infectivity in the supernatants were determined at each time point to estimate the efficiency of viral infectivity grown in the presence of ACV, ASP2151, or PFA. Viral DNA in the cellular fraction contains viral DNA in the virion and replicative form of viral DNA and virus titer in the cellular fraction was not determined.Antiviral assay has been performed to examine the effects of viral DNA synthesis and its related cellular events on the antiviral activity of ASP2151, ACV, and PFA by changing the timing after infection of treatment with antiviral drugs in the infected cultures.

The antiviral drug susceptibility of HSV-infected cells was assessed serially every 2.5 h after infection in order to examine when and how the susceptibility of infected cells to antiviral drugs would change in the replication cycle of HSV infection. Vero cells in 60 mm Petri dishes were infected with HSV-1 at an MOI of 0.01 pfu/cell and incubated for 0, 2.5, 5, 7.5, 10, and 12.5 h at 37°C after 1 h adsorption. Then the infected cells were trypsinized and suspended in 10ml of maintenance medium at 0, 2.5, 5, 7.5, 10, and 12.5h after infection and 0.1 ml was inoculated into Vero cells in 60 mm dishes overlaid with methylcellulose nutrient medium containing drugs. After two days’ incubation, the cells were fixed with 5% formalin and stained with 0.03% methylene blue, and the numbers of plaques were counted. Then the EC50 values of the HSV-1-infected cells for each drug were determined at each time point.Real-time quantitative PCR was performed to quantify the HSV DNA in the collected supernatants and virus-infected HEL cells using Thermal Cycler Dice Real Time System TP800 (Takara Bio Inc., Shiga, Japan). Total DNA was extracted using a High Pure Viral DNA Extraction kit (Roche Applied Science, Mannheim, Germany). The DNA was subjected to a real-time PCR assay using a KAPA SYBR FAST qPCR Kit (Kapa Biosystems, Boston, MA, USA) with primers for HSV TK gene (HSV.SEQ7, 5′- aagccatacccgcttctacaaggc-3′; and HSV.SEQ10, 5′-agggagtggcgcagctgcttc-3′) (Harris et al., 2003). The human GAPDH gene was used as an internal control (forward, 5′- tgtgctcccactcctgatttc-3′; and reverse, 5′-cctagtcccagggtttgatt -3′) (Griscelli et al., 2001).The HSV copy numbers and HSV yields were expressed as the means ± standard deviations of three to four independent wells in each experiment. Comparisons among groups were assessed by two-way repeated-measures ANOVA, followed by the Bonferroni/Dunn method. P <0.05 was considered statistically significant. 3.Results A time-of-drug-addition assay allowed infection at an MOI of 0.4 pfu/cell, and the infected cells were incubated in the presence of an antiviral compound until 48 h after infection. HSV DNA copy numbers were evaluated in the culture supernatant, and cellular fraction and virus yields in the supernatants were determined (Fig. 1). ACV, ASP2151, and PFA significantly inhibited viral DNA copy numbers in the supernatant and cellular fraction and virus yields in the supernatant compared with DMSO-control cultures. ASP2151 and PFA significantly reduced HSV DNA copy numbers in virus-infected cells (Fig. 1A) and the culture supernatant (Fig. 1B) and virus yields (Fig. 1C) compared to ACV. When antiviral drugs were added to the culture medium at 0-4 hpi in 48 h incubation, ASP2151 and PFA reduced HSV DNA copy numbers and virus yields in virus-infected cells to about 1% of those of ACV. Subsequently, ASP2151 and PFA significantly reduced HSV DNA copy numbers as compared with ACV at 4-8 h. PFA significantly reduced intracellular HSV DNA copy number and virus yields but not supernatant HSV DNA more than ASP2151. Thus ASP2151 and PFA inhibited virus replication and reduced yield of infectious virus into culture supernatant more effectively than ACV in the concentrations of 10 x EC50.The addition of antivirals in the early phase of HSV replication produced different profiles of antiviral activity, as shown in Fig. 1. The effect of antivirals on the replication of HSV was evaluated by monitoring viral replication at the indicated times in the presence of drugs. Profiles of inhibition of virus replication by antiviral compounds are shown in Fig. 2. The concentration of antiviral drugs was set at 10 times the EC50 value of ASP2151 and PFA and 10 and 100 times the EC50 value of ACV. Antiviral compounds inhibited virus replication efficiently within 10 h after infection, and ASP2151 and PFA similarly inhibited virus replication as assessed by viral copy number and virus yields for 48 h. ACV at the concentration of 10 x EC50 allowed virus replication up 10 to 100 times of that of ASP2151 or PFA in the supernatant and cellular fraction as assessed by DNA copy number and virus yields, as shown in Fig. 2A, B, and C. However, ACV at 100 x EC50 inhibited viral replication similarly to ASP2151 and PFA. ACV had weaker activity in inhibiting viral replication at the concentration of 10 x EC50 than ASP2151 and PFA. Comparison of 10 x EC50 concentrations among drugs indicated that this weaker inhibition of ACV than ASP2151 and PFA was due to ACV concentration, because the 100 x EC50 concentration showed activity similar to the 10 x EC50 concentration of ASP2151 and PFA. guanosine homopolymeric strings (G-strings) than those from the patients naïve to ACV (Daikoku et al., 2016), indicating virus treated with ACV might have . This suggested the low efficient infectivity of HSV grown in the presence of ACV and the ratios of HSV DNA copy number to infectivity was examined in HSV recovered from the culture supernatants treated with antiviral drugs. ACV at 10 x EC50 allowed virus replication, but the virus titer of the ACV-treated culture supernatant was lower than that in DMSO-treated control culture supernatant in comparison of their respective DNA copy numbers (Fig. 2). Therefore, HSV grown in cultures treated with antiviral agents was characterized by the ratios of HSV DNA copy number to infectivity in the culture supernatant.The profiles of changes in the ratio of HSV DNA copy numbers to infectivity are shown in Fig. 3. HSV replicated in the presence of ASP2151, PFA, and ACV at 10 x EC50 and ACV at 100 x EC50, as shown in Fig. 3, and the profiles of the ratios of HSV copy number to infectivity were similar among the three drugs and time courses in three independent experiments. The ratios of HSV DNA copy number to infectivity were significantly higher in the presence of ASP2151 and ACV at 100 x EC50 than those of ACV at 10 x EC50 and the DMSO control. This indicated that these ratios in treatment with ASP2151 and ACV at 100 x EC50 were produced by remnant viruses in the cultures used for inoculum without replication, because no virus replication was observed as shown in Fig. 2 and should represent the inoculated virus. In contrast, a significant differences in the ratios were observed between HSV replicated in the cells treated with DMSO and ACV at 10 x EC50 (P <0.05), and these ratios were significantly different with those of HSV obtained in treatment with ASP2151 and ACV at 100 x EC50 (P < 0.01). HSV replicated in cultures treated with ACV at 10 x EC50 has significantly higher ratios of HSV DNA copy number to infectivity than HSV treated with DMSO. Virus infectivity produced in the presence of ACV was 3.00 + 1.45 times (n=9) lessefficient per viral DNA copy, and this indicated that ACV-treated virus needed more genomes to attain similar infectivity.Fig. 4 shows the change of EC50 values of HSV-1 infected cells to ACV, ASP2151, and PFA from time 0-12.5 h after infection and the effects of viral DNA synthesis and its related cellular events clearly influenced on the antiviral activity of ASP2151, ACV, and PFA. The EC50 values of ACV increased with time especially after 7.5 h and reached to 8-11 times of the EC50 of 0 h. The EC50 of ASP2151 increased to two to four times of the EC50 of 0 h in the repeated experiments. The EC50 of PFA did not change with a slight increase of 1.25 to 1.70 of the EC50 of 0 h in the repeated experiments. The EC50 increase of infected cells in the time course after infection was preserved in PFA, limited in ASP2151, and much increased for ACV, indicating viral DNA synthesis had little effect on antiviral action of PFA and ASP2151 but influenced that of ACV. This result indicated that susceptibility (EC50) of ACV was greatly influenced by the increase of dGTP for viral DNA synthesis in the late phase of infected cells, in contrast to ASP2151 and PFA. 4.Discussion ACV, ASP2151, and PFA significantly inhibited replication of HSV, and the difference in the mode of antiviral action is shown by the time of their addition during 48 h incubation (Fig. 1). The ACV action was quite different from those of ASP2151 and PFA. ASP2151 and PFA similarly suppressed virus production to undetectable levels in 3 h, while ACV allowed a low level of replication (Fig. 2). ASP2151 allowed a low level of replication at 4 h and later, with increasing replication over time; this may correspond to the beginning of DNA replication, possibly due to the production of large amounts of helicase-primase in the infected cells. This suggests that the ratio of ASP2151 and the helicase-primase molecules may be an important factor in inhibiting viral replication, as was similarly observed in inhibition of viral replication by PFA. ASP2151 and PFA inhibited virus replication and reduced the yield of infectious virus into culture supernatant more effectively than ACV when antiviral compounds were added to the culture media in the early phase of virus replication, although the inhibitory effects of ASP2151, ACV, and PFA on virus replication would be diminished once vigorous viral DNA synthesis begins.ASP2151 inhibits the single-stranded DNA-dependent ATPase, helicase, and primase activities by binding to helicase-primase complex (Biswas et al., 2009; Chono et al.; Chono et al.; James et al.), and PFA inhibits viral DNA polymerase activity through direct binding to the pyrophosphate-binding site (James et al.). Both antivirals directly inhibit viral enzymes. On the other hand, ACV is phosphorylated by HSV TK and cellular kinase, which convert it to the active form, ACV-TP, and then incorporate it into the elongating viral DNA strand, resulting in chain termination (James et al.). Because ACV-TP competes with dGTP to inhibit viral DNA synthesis, the ratio of ACV-TP to dGTP directly affects the effectiveness of ACV in virus replication. Thus, it is reasonable that ASP2151 and PFA, which directly inhibit the viral enzyme, suppressed viral replication more effectively than ACV. The time course of susceptibility change of infected cells showed the ratio of ACV-TP to abundant dGTP directly reduced the effectiveness of ACV in cells with viral DNA synthesis as assessed by the EC50 values and susceptibilities of ASP2151 and PFA were not influenced by viral DNA synthesis in Fig. 4.The drug concentration of ACV, ASP2151, and PFA was 10 x EC50. ACV allowed a low level of replication in its presence, while ASP2151 and PFA inhibited viral replication at thesame 10 x EC50 concentration, as shown in Fig. 2. Because the 100 x EC50 concentration of ACV inhibited viral replication similar to ASP2151 and PFA, this might be due to the different mode of action, and antiviral activity may not reflect well the EC50 determination determined by the plaque reduction assay.We compared the recoveries of HSV replication after removal of ACV and ASP2151, and ASP2151 delayed the recovery of virus replication longer than ACV did (data not shown). However, ACV at 10 x EC50 allowed viral replication, while ASP2151 restricted it to the basal level. Therefore, the recovery of viral replication after drug withdrawal would not be a suitable condition to compare their drug actions, even using the same antiviral potency assessed by 10 x EC50 values.We have analyzed the susceptibility to ACV and the frequency of ACV-resistant mutant in the isolates from the patients naïve to ACV, experienced episodic therapy or suppressive therapy (Daikoku et al., 2016). Susceptibility and frequency of ACV resistant mutants in 104 plaque forming units were not influenced by ACV therapy among three patients groups. Surprisingly the isolates from patients experienced either episodic therapy or suppressive therapy had significantly more frequent mutation in G-strings than those from the patients naïve to ACV. This indicated ACV generated subclinical G-string mutations even during episodic ACV treatment and the generated G-string mutants became additional latent virus for next reactivation. The virus replicated in the presence of ACV had 3 times less efficient infectivity per viral DNA copy than that in the absence of an antiviral agent as shown in Fig. 3, and this may suggest the need for 3 times more genome to attain the same infectivity by the presence of G-string mutation in the genome.Phase II clinical studies with ASP2151 in patients with HSV infections have been completed. In the phase II dose-finding study in patients with recurrent genital herpes, ASP2151 (100, 200, or 400 mg daily for three days, or 1200 mg as a single dose) appears tobe an effective and safe option for treatment of episodes of recurrent genital herpes (Tyring et al.).ASP2151 shows in vitro and in vivo efficacy against HSV-1, HSV-2, and ACV-resistant/TK-deficient virus infection (Himaki et al.; Katsumata et al.) and exhibits a synergistic activity against HSV-1 and HSV-2 with ACV and VACV in vitro and in vivo, respectively (Chono et al.). ASP2151-resistant mutants showed attenuated growth and pathogenicity (Chono et al.). Further, ASP2151 shows efficacy in vitro against varicella-zoster virus (Chono et al., 2010; Chono et al., 2012a; Chono et al., 2013; Himaki et al., 2012). The Phase III clinical trial in herpes zoster patients has been successfully finished.Although in limited conditions, ASP2151 demonstrated a better profile in antiviral activity in during DNA synthesis phase than ACV as an anti-herpesvirus drug and better pharmacokinetics to maintain antiviral serum concentration and effectiveness with once-a-day administration in humans than ACV. Anti-HSV activity of the ASP2151 was not affected by viral DNA synthesis in infected cells compared to ACV as shown in this study, indicating the better efficacy in severe HSV infection where viral DNA synthesis in abundant. ASP2151 will be beneficial as an anti-herpetic drug in addition to ACV, valaciclovir, and famciclovir. Conclusion ASP2151 showed a good profile in the inhibition of viral replication in terms of the appearance and maintenance of antiviral activity and viral DNA synthesis did not influenced anti-HSV activity of ASP2151 compared to ACV. ASP2151 directly inhibits helicase-primase, while ACV-TP phosphorylated by viral TK competes with abundant dGTP in viral DNA synthesis. The results of this study show that ASP2151 reduces HSV DNA synthesis and infectious virus to lower levels than the equivalent Amenamevir concentration of ACV during the early stages of replication. Thus ASP2151 had a better profile as an anti-herpetic drug than acyclovir in this study.