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. Author manuscript; available in PMC: 2021 Mar 19.
Published in final edited form as: Antiviral Res. 2020 Jan 20;175:104713. doi: 10.1016/j.antiviral.2020.104713

Biosynthesis and Half-Life of MBX-2168-Triphosphate in Herpes Virus-Infected Cells

HANNAH E SAUER a, MARIE L NGUYEN b, JOHN D WILLIAMS c, TERRY L BOWLIN c, BRIAN G GENTRY a,*
PMCID: PMC7976169  NIHMSID: NIHMS1551642  PMID: 31968222

Abstract

The third generation of methylenecyclopropane nucleoside analogs (MCPNAs) elicit an anti-viral effect against all three sub-classes of herpes viruses without inducing cytotoxicity in vitro. It has been previously established that the mechanism of action of MCPNAs is similar to that of ganciclovir (GCV) or acyclovir (ACV). However, the activation of MBX-2168, a third generation MCPNA, involves additional and unique enzymatic steps and this process has not been examined in virus-infected cells. To that end, herpes virus-infected cells were incubated with MBX-2168, synguanol, GCV, or ACV. Incubation of HCMV-infected cells with five times the EC50 of MBX-2168 (4.0 μM), synguanol (10.5 μM), or GCV (25 μM) resulted in a time-dependent increase in triphosphate accumulation reaching a maximum of 48.1 ± 5.5, 45.5 ± 2.5, and 42.6 ± 3.7 pmol/106 cells at 120 hours, respectively. Additionally, half-lives of these compounds were similar in HCMV-infected cells (GCV-TP = 25.5 ± 2.7 hours; MBX-2168-TP/synguanol-TP = 23.0 ± 1.4 hours). HSV-1-infected cells incubated with five times the EC50 of MBX-2168 (33.5 μM) or ACV (5.0 μM) demonstrated a time-dependent increase in triphosphate levels reaching a maximum of 12.3 ± 1.5 and 11.6 ± 0.7 pmol/106 cells at 24 hours, respectively. ACV-TP and MBX-2168-TP also had similar half-lives under these conditions (27.3 ± 4.8 hours and 22.2 ± 2.2 hours, respectively). We therefore conclude that although MBX-2168 does not follow the classical route of nucleoside analog activation, the metabolic profile of MBX-2168 is similar to other nucleoside analogs such as GCV and ACV that do.

Keywords: methylenecyclopropane nucleoside analogs, cytomegalovirus, herpes simplex virus, drug metabolism

1. INTRODUCTION

The herpes family of viruses is characterized by their ability to establish latency and reactivate upon immune system dysfunction or inactivation (Pellett and Roizman, 2013). The pathogenicity of the herpes viruses ranges in severity, but are generally of significant concern for patients with immature or suppressed immune systems such as those with AIDS, organ transplants, and receiving cancer chemotherapy. Herpes simplex virus type-1 (HSV-1) a member of the alpha herpesviridae subfamily, affects up to 80% of the worldwide population (Tognarelli et al., 2019). HSV-1 infection in its active form is commonly referred to as “cold sores”, but is an example of one herpes virus that can have serious (sometimes fatal) effects, including neuralgias and meningoencephalitis, in individuals with suppressed immune systems (Whitley et al., 1998). Human cytomegalovirus (HCMV), a member of the beta herpesviridae subfamily, affects up to 90% of the adult population worldwide and can cause severe disease (encephalitis, retinitis, and enteritis) in immunocompromised patients (Landolfo, 2003). In addition, HCMV is the most common congenital infection in the industrialized world with manifestations in infants including hearing loss and mental development impairment (Kenneson and Cannon, 2007).

Primary pharmacotherapy options for the treatment of systemic herpes virus infections include the nucleoside analogs acyclovir (ACV) and ganciclovir (GCV) (Fig 1) (Britt and Prichard, 2018; James and Prichard, 2014). The mechanisms of action for ACV (the primary pharmacotherapy option for systemic HSV-1 infections) and GCV (the primary option for systemic HCMV infections) are similar; namely, initial phosphorylation by a virus specific enzyme (HSV thymidine kinase, HCMV pUL97), further phosphorylation to a triphosphate by endogenous enzymes, and direct inhibition of the viral polymerase. In addition, GCV can compete with the endogenous deoxyribonucleoside triphosphates for incorporation into replicating viral DNA resulting in premature chain termination (Razonable, 2011). However, these pharmacotherapies suffer from two major drawbacks. First, due to the nature of administration (life-long adherence due to recurrence of infection upon cessation of therapy), the development of viral strains with decreased susceptibility to drug is common (Andrei and Snoeck, 2013; Lurain and Chou, 2010). In addition and since these drugs share the same viral target, the development of cross-resistance or resistance to one drug conferring resistance to another, is an increasing concern (Drew et al., 2001). The second drawback is a high incidence rate of adverse effects from those drugs used to treat systemic HCMV infections. GCV has a high incidence of neutropenia (~30% of patients) (Crumpacker, 1996). Furthermore, for GCV-resistant HCMV infections, administration of cidofovir or foscarnet results in ~50% incidence rate of nephrotoxicity (Lea and Bryson, 1996; Tan, 2014). Combined, these issues demonstrate a pressing need for the development of therapy options that increase the therapeutic index and/or can be utilized against drug-resistant viral isolates.

Figure 1.

Figure 1.

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Structures of acyclovir (ACV), ganciclovir (GCV), synguanol, and MBX-2168.

The methylenecyclopropane nucleoside analogs (MCPNAs) are one promising class of drugs that have demonstrated greater efficacy when compared to the current treatments for HCMV without any observed increase in cytotoxicity. A first generation compound, synguanol (Fig 1, EC50 vs HCMV = 2.1 μM), demonstrated a modest increase in antiviral activity when compared to GCV (EC50 = 7.4 μM) (Qiu et al., 1998). Filociclovir (FCV), a second generation MCPNA currently in clinical trials, demonstrated 10-fold greater efficacy against HCMV (EC50 = 0.46 μM) when compared to GCV (EC50 = 4.1 μM) (Zhou et al., 2004). However, FCV primarily elicits an effect against the β-herpes subfamily with limited efficacy against α- and γ-herpes viruses (Kern et al., 2005; Zhou et al., 2004). MBX-2168 (Fig 1), a prototypical third generation MCPNA, elicits an effect against a broad range of herpes viruses including HCMV (EC50 = 0.8 μM) and HSV (EC50 = 6.7 μM (Prichard et al., 2013).

The mechanism of action for the MCPNAs is similar to that of the nucleoside analogs GCV and ACV (Chen et al., 2016; Gentry et al., 2011; Gentry et al., 2010; Gentry et al., 2013; Kern et al., 2005; Komazin-Meredith et al., 2014). There are, however, nuances with the third generation of MCPNA that distinguish them from both the classical nucleoside analogs (GCV and ACV) and the previous generations of MCPNAs. First, the initial phosphorylation either in part or in whole is facilitated by the cellular enzyme TAOK3 (Komazin-Meredith et al., 2015). And second, the conversion of MBX-2168 to a triphosphate undergoes an additional step – removal of the butyl ether at the 6-position of the guanine ring, essentially converting MBX-2168-MP into synguanol-MP by the enzyme adenosine deaminase-like protein 1 (Vollmer et al., 2019). Although these enzymatic conversions have been established, they have not been observed in virus-infected cells. Therefore, the goal of this study is to examine the biosynthesis and half-life of MBX-2168-triphosphate (-TP) in both HCMV- and HSV-infected cells.

2. METHODS

2.1. Viral strains and chemicals.

HCMV Towne was kindly provided by M. F. Stinski, University of Iowa. KOS1.1 strain of HSV was generously provided by Dr. John Blaho of Mount Sinai School of Medicine (New York, NY). GCV and ACV were purchased from Millipore Sigma (St. Louis, MO). Synguanol was kindly provided by Dr. Jiri Zemlicka (Karmanos Cancer Center, Wayne State University, Detroit, MI). The synthesis of MBX-2168 was described previously (Prichard et al., 2013). 8-3H-GCV (19 Ci/mmol), 8-3H-ACV (3.2 Ci/mmol), 8-3H-synguanol (3.1 Ci/mmol), and 8-3H-MBX-2168 (3.0 Ci/mmol) were purchased from Moravek Biochemicals (Brea, CA).

2.2. Cell culture procedures.

Human foreskin fibroblasts (HFF) were grown in minimal essential medium with Earle’s salts, supplemented with penicillin and streptomycin, and 10% fetal bovine serum. Vero cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with penicillin and streptomycin, Fungizone, and 5% fetal bovine serum. Cells were grown at 37°C in a humidified atmosphere of 3–5% CO2 and 97–95% air and were regularly passaged at 1:2 dilutions (HFF) or 1:2 – 1:20 dilutions (Vero) using conventional procedures with 0.05% trypsin–0.02% EDTA–HEPES-buffered saline.

2.3. Biosynthesis of nucleoside analog triphosphate in herpes virus-infected cells.

HFF cells were seeded at 250,000 cells per well in 6-well cluster dishes and allowed to reach 80–90% confluency overnight. The following day, cells were infected at an MOI of >5 plaque forming units (pfu) per cell with HCMV Towne. Two hours post-infection, 25 μM GCV (~2% 3H-), 10.5 μM synguanol (~4% 3H-), or 4.0 μM MBX-2168 (~1% 3H-) was added to the cells. At designated times, the cells were removed using trypsin, lysed with water, and proteins were precipitated using perchloric acid (0.4 N final concentration). Samples were then neutralized using 10.0 N potassium hydroxide and stored at −20°C until analysis by high pressure liquid chromatography (HPLC).

Vero cells grown in Dublecco’s modification of Eagle’s medium were seeded at 800,000 cells per well in 6-well cluster dishes and allowed to reach 80–90% confluency overnight. The following day, cells were infected at an MOI of >5 PFU/cell with HSV-1 KOS1.1. One hour post-infection, 5.0 μM ACV (~4% 3H-) or 33.5 μM MBX-2168 (~1% 3H-) were added to the cells. At designated times, cells were processed using the same procedure described above.

2.4. Half-life of nucleoside analog triphosphate in herpes virus-infected cells.

HFF cells were seeded at 250,000 cells per well in 6-well cluster dishes and allowed to reach 80–90% confluency overnight. The following day, cells were infected at an MOI of >5 PFU/cell with HCMV Towne. Two hours post-infection, 25 μM GCV (~2% 3H-) or 4.0 μM MBX-2168(~1% 3H-) was added to the cells. Following five days of drug incubation, media containing drug was removed and replaced with fresh media without drug. Samples were collected at the time of and at designated times following media replacement and processed as described above.

Vero cells were seeded at 800,000 cells per well in a 6-well cluster dish and allowed to reach 80–90% confluency overnight. The following day, cells were infected at an MOI of >5 PFU/cell with HSV-1 KOS1.1. One hour post-infection, 5.0 μM ACV (~4% 3H-) or 33.5 μM MBX-2168(~1% 3H-) were added to the cells. Following 24 hours of incubation with drugs, media containing drug was replaced with fresh media without drug. Samples were collected at the time of and at designated times following media replacement and processed as described above.

2.5. Reverse phase HPLC.

Synguanol, MBX-2168, and their respective phosphorylated derivatives were separated using reverse-phase HPLC [Agilent Technologies, Inc. (Santa Clara, CA) 1200 Infinity Series]. Prior to injection, each sample was centrifuged at 13,300 rpm for 10 minutes to remove particulate matter. Samples were loaded onto a 10 μm Alphabond C18 300 × 3.9 mm reverse-phase column (Alltech, Deerfield, IL) at a flow rate of 1.0 ml/min. Separation was achieved by eluting with 1.0 mM ammonium phosphate (pH 3.0) and 100% methanol (95% ammonium phosphate:5% methanol initial with a linear gradient of 25% methanol over thirty minutes, followed by isocratic conditions of 25% methanol, 75% ammonium phosphate for 10 minutes). One-minute fractions were collected and tritium quantified by liquid scintillation spectrometry using a Tricarb 2100 TR Liquid Scintillation Analyzer [PerkinElmer (Waltham, MA)].

2.6. Strong anion exchange HPLC.

GCV, ACV, and their respective phosphorylated derivatives were separated using strong anion exchange HPLC [Agilent Technologies, Inc. (Santa Clara, CA) 1200 Infinity Series]. Prior to injection, each sample was centrifuged at 13,300 rpm for 10 minutes to remove particulate matter. Samples were loaded onto a 5 μm Hypersil 250 × 4.6 mm strong anion exchange column (Thermo Scientific, Waltham, MA) at a flow rate of 1.0 ml/min. Separation was achieved by eluting with 10 mM ammonium phosphate (pH 3.0) and 500 mM ammonium phosphate (pH 3.0) (10 mM ammonium phosphate isocratic conditions for 10 minutes followed by a 60% gradient of 500 mM ammonium phosphate for 30 minutes). One minute fractions were collected and tritium quantified by liquid scintillation spectrometry using a Tricarb 2100 TR Liquid Scintillation Analyzer [PerkinElmer (Waltham, MA)].

2.7. Data analysis.

The concentration of nucleoside analog triphosphate for each drug was calculated based upon the amount of label in the HPLC effluent fractions corresponding to a known or estimated position of the metabolite, the specific activity of the tritium drug solutions, and the cell count for the samples. The results were graphed using Prism (version 5.0; GraphPad Software, San Diego, CA) to determine standard deviations, linear regressions, statistical significance, and areas under the curve.

3. RESULTS

3.1. Biosynthesis of MCPNA-TP and GCV-TP in HCMV-infected cells.

We have previously examined the phosphorylation of a second generation MCPNA (FCV) to a triphosphate in HCMV-infected cells (Gentry and Drach, 2014). In contrast, using enzymatic approaches we recently have determined that the phosphorylation of MBX-2168 (a third generation MCPNA) to a triphosphate has two unique metabolic steps – initial phosphorylation by an endogenous cellular enzyme (Komazin-Meredith et al., 2015) and the removal of an ether moiety at the purine 6-position (Vollmer et al., 2019). We now have extended these studies and examined the phosphorylation profile of MBX-2168 in HCMV-infected cells. HFF cells infected with HCMV Towne were incubated with equipotent concentrations of GCV (25.0 μM) or MBX-2168 (4.0 μM) and cell extracts were analyzed for presence of nucleoside analog phosphates. Incubation with 4.0 μM MBX-2168 resulted in a time-dependent increase in triphosphate levels reaching a maximum of 48.1 ± 5.5 pmol/106 cells at 120 hours (Fig 2a; Table 1). In addition and similar to what we previously observed (Gentry and Drach, 2014), HCMV-cells incubated with 25.0 μM GCV demonstrated a time-dependent increase in triphosphate levels reaching a maximum of 42.6 ± 2.5 pmol/106 cells at 120 hours (Fig 2a; Table 1). The results demonstrated that the accumulation MBX-2168-TP was not statistically different from that of GCV-TP under these experimental conditions. Additionally, no detectable levels of mono- or diphosphates were observed indicating that the rate-limiting step is the initial phosphorylation.

Figure 2.

Figure 2.

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a. Biosynthesis of GCV-TP, MBX-2168-TP, and Synguanol-TP in HCMV-infected HFF cells. HCMV-infected cells were incubated with five times the EC50 of either GCV (25 μM), MBX-2168 (4.1 μM), or synguanol (10.5 μM). Samples were taken and processed at designated times following the addition of drugs. Maximum accumulations of GCV-TP (▲; 42.6 ± 3.7 pmol/106 cells), MBX-2168-TP (■; 48.1 ± 5.5 pmol/106 cells), and synguanol-TP (◆; 45.5 ± 2.5 pmol/106 cells) were equivalent. b. Depletion of GCV-TP and MBX-2168-TP in HCMV-infected HFF cells. HCMV-infected cells were incubated as above with either GCV (25 μM) or MBX-2168 (4.1 μM). After five days drug-containing media was replaced with fresh media without drug. Samples were taken and processed at designated time points following drug removal. Both compounds demonstrated first order kinetics and half-lives were calculated accordingly. The half-lives of GCV-TP (▲) and MBX-2168-TP (■) were not statistically different, 25.5 ± 2.7 hours and 23.0 ± 1.4 hours, respectively.

Table 1.

Comparison of MBX-2168-TP, Synguanol-TP, and GCV-TP Metabolism in HCMV-Infected Cells.

Compound Peak Triphosphate Level Half-Life (t1/2) Area Under the Curve (AUC)
(pmol/106 cells)* (Hours) (pmol·hrs/106 cells)‡
MBX-2168-TP 48.1 ± 5.5 23.0 ± 1.4 3830 ± 450
Synguanol-TP 45.5 ± 2.5 ND ND
GCV-TP 42.6 ± 3.7 25.5 ± 2.7 3670 ± 380
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*

Mean ± standard deviation from at least three experiments.

‡

Value calculated as a result of combining the data from the triphosphate biosynthesis and half-life studies.

ND – Not Determined

It has previously been determined that MBX-2168-MP is enzymatically converted to synguanol-MP by ADAL1 (Vollmer et al., 2019). Furthermore, subsequent phosphorylation of MBX-2168 into a triphosphate results in a compound identical to the respective triphosphate of synguanol, indicating that the parent compounds MBX-2168 and synguanol have structurally identical active metabolites. Due to this relationship, the biosynthesis of MBX-2168-TP was compared to the biosynthesis of synguanol-TP in HCMV-infected cells. Incubation of HCMV-infected HFF cells with 10.5 μM synguanol demonstrated a time-dependent increase in synguanol-TP reaching a maximum of 45.5 ± 2.5 pmol/106 cells at 120 hours (Fig 2a; Table 1). Under these experimental conditions (equivalently effective concentrations; 5xEC50) there is no statistical difference in the accumulation of synguanol-TP and MBX-2168-TP. In addition, no other metabolites of MBX-2168, GCV, or synguanol were detected under these experimental conditions.

Uninfected HFF cells demonstrated no measurable levels of synguanol or GCV phosphates indicating that the presence of the virus and specifically the viral kinase pUL97 is necessary for activation of synguanol and GCV (Gentry et al., 2010). However, uninfected HFF cells did produce MBX-2168-TP (18.5 ± 2.2 pmol/106 cells at 120 hours) albeit at 2.5 times lower final concentration (Table 2). This indicates that endogenous cellular enzymes can phosphorylate MBX-2168 to a triphosphate in the absence of virus (Komazin-Meredith et al., 2015).

Table 2.

Comparison of MBX-2168-TP in herpes virus infected and uninfected cells.

Cell Type Virus Peak Triphosphate Level
(pmol·hrs/106 cells)*
HFF HCMV Towne 48.1 ± 5.5
None 18.5 ± 2.2
Vero HSV KOS-1.1 12.3 ± 1.5
None 15.9 ± 3.0
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*

Mean ± standard deviation from at least three experiments.

3.2. Half-life of MBX-2168-TP and GCV-TP in HCMV-infected cells.

Drug half-life is another important component of drug efficacy because it determines the length of time that virus is exposed to the active compound. Therefore the half-life of MBX-2168-TP and GCV-TP were measured in HCMV-infected cells following 5 days of exposure to 4.0 μM MBX-2168 or 25.0 μM GCV. The results demonstrated first-order kinetics for both drugs and half-lives were calculated from the data (Fig 2b). MBX-2168-TP and GCV-TP demonstrated half-lives of 23.0 ± 1.4 and 25.5 ± 2.7 hours, respectively (Table 1).

3.3. Biosynthesis of MCPNA-TP and ACV-TP in HSV-infected cells.

MBX-2168 demonstrates increased anti-viral potency compared to second generation MCPNAs. As such, we examined the biosynthesis of MBX-2168-TP in an α-herpes virus, HSV-1. Vero cells infected with the KOS-1.1 strain of HSV-1 were incubated with equipotent concentrations of ACV (5.0 μM) or MBX-2168 (33.5 μM) and cell extracts were analyzed for presence of nucleoside analog phosphates. Incubation of HSV-infected Vero cells with MBX-2168 resulted in a time-dependent increase in triphosphate level with a maximum 12.3 ± 1.5 pmol/106 cells at 24 hours (Fig 3a; Table 3). HSV-infected cells incubated with ACV demonstrated a similar time-dependent increase in triphosphate levels, reaching a maximum of 11.6 ± 0.7 pmol/106 cells at 24 hours (Fig 3a; Table 3). Uninfected Vero cells demonstrated no measurable level of ACV phosphates, confirming that the presence of an activating viral kinase (HSV thymidine kinase) is necessary for the initial phosphorylation of ACV in HSV-infected cells (James and Prichard, 2014). However, uninfected Vero cells did demonstrate levels of MBX-2168-TP (15.9 ± 3.0 pmol/106 cells at 24 hours), a level not statistically different from levels of MBX-2168-TP in HSV-infected cells (Table 2). This indicates that unlike ACV, MBX-2168 does not require a viral protein for activation (initial phosphorylation). Again, no mono- or diphosphate metabolites were detected, indicating that initial monophosporylation is the rate-limiting step. Additionally, no other metabolites of MBX-2168 or ACV were detected under the conditions used for these experiments.

Figure 3.

Figure 3.

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a. Biosynthesis of ACV-TP and MBX-2168-TP in HSV-infected Vero cells. HSV-infected cells were incubated with five times the EC50 of either ACV (5.0 μM) or MBX-2168 (33.5 μM). Samples were taken and processed at designated times following the addition of drug. Time-dependent increases were observed in ACV-TP (●) and MBX-2168-TP (■); maximum concentrations were 11.6 ± 0.7 and 12.3 ± 1.5 pmol/106 cells, respectively. b. Depletion of ACV-TP and MBX-2168-TP in HSV-infected Vero cells. HSV-infected cells were incubated as above with either ACV (5.0 μM) or MBX-2168 (33.5 μM). After 24 hours drug-containing media was replaced with fresh media without drug. Samples were taken and processed at designated time points following drug removal. Both compounds demonstrated first order kinetics and half-lives were calculated accordingly. The half-lives of ACV-TP (●) and MBX-2168-TP (■) were not statistically different, 22.2 ± 2.2 hours and 27.3 ± 4.8 hours, respectively.

Table 3.

Comparison of MBX-2168-TP and ACV-TP Metabolism in HSV-Infected Cells.

Compound Peak Triphosphate Level Half-Life (t1/2) Area Under the Curve (AUC)
(pmol/106 cells)* (Hours) (pmol·hrs/106 cells)‡
MBX-2168-TP 12.3 ± 1.5 22.2 ± 2.2 634 ± 105
ACV-TP 11.6 ± 0.7 27.3 ± 4.8 359 ± 91
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*

Mean ± standard deviation from at least three experiments.

‡

Value calculated as a result of combining the data from the triphosphate biosynthesis and half-life studies.

3.4. Half-life of MBX-2168-TP and ACV-TP in HSV-infected cells.

Half-lives of MBX-2168-TP and ACV-TP were measured in HSV-infected cells following 24 hours of exposure to 33.5 μM MBX-2168 or 5.0 μM ACV. The results demonstrated first-order kinetics for both drugs (Fig 3b); half-lives were calculated to be 22.2 ± 2.2 and 27.3 ± 4.8 hours, respectively (Table 2).

3.5. Exposure of nucleoside analog triphosphates to HSV- and HCMV-infected cells.

Although peak accumulation of triphosphate is valuable information for comparing the biosynthesis of the active compounds, area under the curve (AUC) more accurately reflects the exposure of virus to activated drug. For HCMV-infected cells, the results demonstrated that under the conditions used in these experiments the areas under the curve (AUC) for MBX-2168-TP and GCV-TP are 3830 ± 450 and 3670 ± 380 pmol·hours/106 cells, respectively (Table 1) and are not statistically different. AUC for MBX-2168-TP and ACV-TP in HSV-infected cells are 634 ± 105 and 359 ± 91 pmol·hours/106 cells, respectively (Table 2). These results are statistically different indicating that the exposure of HSV-infected cells to MBX-2168-TP is greater than that of ACV-TP under equivalently effective concentration conditions (five times the EC50).

4. DISCUSSION

We have previously examined the biosynthesis and half-lives of FCV-TP, a second generation MCPNA, and GCV-TP in HCMV-infected cells (Gentry and Drach, 2014). Under equivalently effective concentrations of five times the EC50, HCMV-infected cells were exposed to FCV-TP to a much greater extent than to GCV-TP. When compared to this study, there is no statistically significant difference in the accumulation of GCV-TP in HCMV-infected cells, demonstrating consistency between the two studies. The AUC in HCMV-infected HFF cells (Table 1) indicates equal exposure to triphosphate and thus equal potencies for GCV-TP and MBX-2168-TP. Conversely, the aforementioned increased accumulation of FCV-TP under these conditions demonstrates its lesser potency as an active metabolite. This confirms that the overall potency of the parent compounds (CPV > MBX-2168 > GCV) is due primarily to selective activation rather than any increase in efficacy of their respective active triphosphates.

While the mechanism of action of MBX-2168 is similar to that of other nucleoside analogs (phosphorylation into a triphosphate which results in inhibition of viral DNA synthesis), it involves additional metabolic conversions unlike the current standards of pharmacotherapy (GCV and ACV) and the previous generations of MCPNAs (Komazin-Meredith et al., 2015; Vollmer et al., 2019). These additional steps include the removal of the butyl ether at the 6 position of the guanine ring, converting MBX-2168-MP to a compound chemically identical to the monophosphate of synguanol, a first generation MCPNA. Our results indicate that these unique structural anomalies optimize the selectivity of MBX-2168 and its triphosphate when compared to previous compound generations. The structure of FCV, a second generation MCPNA, consists of two methyl hydroxyls attached to a cyclopropane moiety, whereas synguanol and MBX-2168 contain one (Fig 1). While the presence of an additional methyl hydroxyl incurs increased affinity for pUL97 in HCMV-infected cells, our results also demonstrate that it decreases the potency of the triphosphate metabolite for the viral DNA polymerase (pUL54) (Gentry and Drach, 2014). Conversely, our current results indicate an increased potency of synguanol-TP for the viral DNA polymerase when compared to FCV-TP, but a lower potency of parent compound when compared to FCV (Qiu et al., 1998; Zhou et al., 2004) indicates a decreased affinity for the enzyme responsible for catalyzing the initial phosphorylation. Our current results also demonstrate that MBX-2168-TP, which is chemically identical to synguanol-TP, has increased potency for the viral polymerase when compared to FCV-TP (Gentry and Drach, 2014). And as a parent compound, MBX-2168 has potency comparable to that of FCV (Prichard et al., 2013; Zhou et al., 2004) which is due primarily to increased phosphorylation to a monophosphate when compared to synguanol. Thus, MBX-2168 appears to have the advantages from each of the first two generations of MCPNAs (namely synguanol and FCV).

The detection of MBX-2168-TP in uninfected HFF and Vero cells indicate an unique anomaly in the mechanism of action of MBX-2168. In uninfected HFF cells, MBX-2168-TP accumulated to 18.5 ± 2.2 pmol/106 cells, a concentration 2.5 times lower than that of HCMV-infected cells (48.1 ± 5.5 pmol/106 cells) (Table 2). The difference in accumulation can be attributed to the absence of the viral protein kinase pUL97, which also phosphorylates MBX-2168 to a monophosphate (Komazin-Meredith et al., 2014). However in uninfected Vero cells, MBX-2168-TP accumulated to a level similar to that observed in HSV-infected cells. Previous studies have demonstrated that HSV thymidine kinase activity is not an essential part of the mechanism of action of MBX-2168 (Komazin-Meredith et al., 2015). That same study indicates that the endogenous cellular enzyme TAOK3 can phosphorylate MBX-2168 to a monophosphate. Therefore, the presence of HSV is not essential for the biosynthesis of MBX-2168-TP since an endogenous cellular enzyme can perform the initial phosphorylation. Consistent with these studies, our results demonstrate equivalent production of MBX-2168-TP in both HSV-infected and uninfected cells (12.3 ± 1.5 and 15.9 ± 3.0 pmol/106 cells, respectively) (Table 2).

Phosphorylation to a triphosphate in uninfected cells represents a new mode of activation/action for nucleoside analogs to combat herpes viral infections, but something that has been previously demonstrated with nucleoside analogs used for the treatment of retroviruses (Arts and Hazuda, 2012). In addition, MBX-2168 has demonstrated activity against GCV- or ACV-resistant herpes virus infections when the mutation that causes resistance occurs in the activating enzyme rather than the target protein since formation of the active compound (-TP) can still occur (Komazin-Meredith et al., 2015). This suggests that the ability of MBX-2168 to overcome resistance when the mutation occurs in the enzyme of activation rather than the target protein is due to the endogenous cellular activation of the compound. Consistent with this, we demonstrated herein the ability of uninfected cells to phosphorylate MBX-2168 to a triphosphate (Table 2). As such, further analysis of triphosphate formation in uninfected and mutant-infected cells is warranted given that this drug could be utilized for the treatment of drug-resistance viral infections.

The accumulation of MBX-2168-TP in uninfected cells also indicates a different mechanism of selectivity from that of GCV. For GCV, the selective antiviral effect is due to a large degree from selective phosphorylation of the compounds in virus-infected cells (Biron, 2006; Britt and Prichard, 2018). If phosphorylated to a monophosphate, endogenous cellular enzymes can further phosphorylate the nucleoside analog to a triphosphate and it can elicit a cytotoxic effect. In fact, this is the basis of an experimental type of cancer chemotherapy – suicide gene therapy where an enzyme that can monophosphorylate the compound results in an anti-cancer effect (Rubsam et al., 1998). Conversely, MBX-2168 does not need a viral specific enzyme to catalyze the initial phosphorylation (Komazin-Meredith et al., 2015). As such, uninfected cells produce levels of active triphosphate sufficient to elicit an anti-viral effect but do not display any observable level of cytotoxicity. Thus, we speculate that the mode of selective activity for MBX-2168 is at the level of the target (the MCPNA triphosphates are recognized substrates by the viral polymerase, but not the endogenous mammalian DNA polymerases) rather than selective activation/monophosphorylation in herpes virus infected cells. We would further hypothesize that based on the structural similarities between all three generations of MCPNAs, that selectivity for the early generations of MCPNAs is a result of both activation and preferential affinity for target.

The half-life and biosynthesis of ACV-TP in HSV-infected cells has been previously examined. When HSV-infected cells (MOI >5 pfu) were incubated with 5.0 μM ACV, Elion et al. reported triphosphate accumulation reaching a peak of 3.5 pmol/106 cells at 7 hours (Elion et al., 1977). These results are consistent with our findings – the measured level of ACV-TP in HSV-infected cells at 8 hours was 3.8 ± 0.3 pmol/106 cells and interpolation of our results for 7 hours demonstrates a level similar to that of the Elion study. In another study, Furman et al. demonstrated a time-dependent increase in triphosphate accumulation reaching 20.1 pmol/106 cells at 7 hours (Furman et al., 1981). This value differs from what we report and can be mainly attributed to the drug concentration used in each of the studies. Furman et al. incubated HSV-infected cells with 100 μM ACV or 100 times the reported EC50 of 1.0 μM. Conversely, in our current study we administered a concentration of five times the EC50 for all drugs, in this case, 5.0 μM ACV.

When comparing the biosynthesis of ACV-TP and MBX-2168-TP in HSV-infected cells, a notable delay in production of triphosphate is observed for ACV, but not MBX-2168 (Fig 3). The explanation for this difference is simple – each compound in initially phosphorylated by a different enzyme. Early-phase enzymes, such as HSV-TK are not expressed until 2 hours post infection and not fully expressed until 3 to 4 hours into the 24-hour viral replication cycle of HSV (Pellett and Roizman, 2013). Thus, when we examine the biosynthesis of ACV-TP, there is an approximate 2 hour delay due to the lack of enzyme necessary for the initial phosphorylation step. Conversely, it has been previously demonstrated that a constitutively expressed endogneous enzyme, TAOK3 is able to convert MBX-2168 into a monophosphate upon exposure to the compound (Komazin-Meredith et al., 2015). Thus the biosynthesis of MBX-2168-TP occurs immediately upon exposure of HSV-infected cells to drug.

The results herein indicate that accumulation of MBX-2168-TP is similar to that of GCV-TP in HCMV-infected cells. Given its greater efficacy as a parent compound with no observed increase in toxicity (Prichard et al., 2013), its viability as a pharmacotherapy option for the treatment of systemic HCMV infection is promising. In addition, the antiviral activity of MBX-2168 has been demonstrated not only in HCMV and HSV, but also HHV-6, indicating its potential for use against both α and β subfamilies of the herpes family (Prichard et al., 2013). Furthermore, the unique mode of activation in HSV-infected cells (activation by TAO3K) (Komazin-Meredith et al., 2015) offers a promising therapy option for HSV resistant to currently available pharmcotherapies, particularly when resistance is due to mutations in the virally encoded thymidine kinase. As such, MBX-2168 continues to be a compound of interest for the treatment of systemic herpes-virus infections and further development of this class of compounds is warranted.

Highlights:

  • Incubation of MBX-2168 or GCV (5xEC50) in HCMV-infected cells resulted in similar triphosphate accumulation

  • Incubation of MBX-2168 or ACV (5xEC50) in HSV-infected cells resulted in similar triphosphate accumulation

  • Half-life of triphosphates (GCV, ACV, or MBX-2168) were all approximately 24 hrs and independent of cell type

  • Despite non-classical route of activation, MBX-2168 has similar intracellular activation kinetics when compared to GCV/ACV

5. ACKNOWLEDGEMENTS

This work was supported by the Iowa Space Grant Consortium (NASA grant number NNX10AK63H), funds from Drake University (Drake Undergraduate Science Collaborative Institute), and the NIH (R44 AI082799, R43 AI082799).

Footnotes

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