6-Thioguanine Inhibits Herpes Simplex Virus 1 Infection of Eyes.

Deyan Chen,Jing Wu,Wenyuan Ma,Qiao You,Fang Zhang,Ye Liu,Zhiwei Wu,Clinton J Jones

doi:10.1128/spectrum.00646-21

Abstract

ABSTRACTHerpes simplex virus 1 (HSV-1) infects eye corneal tissues leading to herpetic stromal keratitis (HSK), which is one of the leading causes of blindness. Here in our study, we found that 6-thioguanine (6-TG), a once clinically approved medication for child acute myelogenous leukemia, inhibited multiple strains of HSV-1 infection in vitro and in vivo. 6-TG is more potent than acyclovir (ACV) and ganciclovir (GCV), with the 50% inhibitory concentration (IC50) of 6-TG at 0.104 μM with high stimulation index (SI) (SI = 6,475.48) compared to the IC50 of ACV at 1.253 μM and the IC50 of GCV at 1.257 μM. In addition, 6-TG at 500 μM topically applied to the eyes with HSV-1 infection significantly inhibits HSV-1 replication, alleviates virus-induced HSK pathogenesis, and improves eye conditions. More importantly, 6-TG is effective against ACV-resistant HSV-1 strains, including HSV-1/153 and HSV-1/blue. Knockdown of Rac1 with small interfering RNA (siRNA) negatively affected HSV-1 replication, suggesting that Rac1 facilitated HSV-1 replication. Following HSV-1 infection of human corneal epithelial cells (HCECs), endogenous Rac1 activity was upregulated by glutathione S-transferase (GST) pulldown assay. We further found that Rac1 was highly expressed in the corneal tissue of HSK patients compared to normal individuals. Mechanistic study showed that 6-TG inhibited HSV-1 replication by targeting Rac1 activity in HSV-1 infected cells, and the Rac1 is critical in the pathogenesis of HSK. Our results indicated that 6-TG is a promising therapeutic molecule for the treatment of HSK.IMPORTANCE We reported the discovery of 6-TG inhibition of HSV-1 infection and its inhibitory roles in HSK both in vitro and in vivo. 6-TG was shown to possess at least 10× more potent inhibitory activity against HSV-1 than ACV and GCV and, more importantly, inhibit ACV/GCV-resistant mutant viruses. Animal model studies showed that gel-formulated 6-TG topically applied to eyes locally infected with HSV-1 could significantly inhibit HSV-1 replication, alleviate virus-induced HSK pathogenesis, and improve eye conditions. Further study showed that HSV-1 infection upregulated Rac1 expression, and knockdown of Rac1 using siRNA markedly restricted HSV-1 replication, suggesting that Rac1 is required for HSV-1 replication. In addition, we also documented that Rac1 is highly expressed in corneal tissues from HSK patients, indicating that Rac1 is associated with HSK pathogenesis. In view of the high potency of 6-TG, low cytotoxicity, targeting a distinct therapeutic target, we suggest that 6-TG is a potential candidate for development as a therapeutic agent for HSK therapy.

Highlights

Herpes simplex virus 1 (HSV-1) infects eye corneal tissues leading to herpetic stromal keratitis (HSK), which is one of the leading causes of blindness
We determined cytotoxicity of 6-TG on a human corneal epithelial cells (HCECs) line and observed that 20% cell death was induced by 50 mM 6-TG at 24 h, and the 50% cytotoxic concentration (CC50) was 700 6 16.3 mM (Fig. 1b). 6-TG inhibited HSV-1 in a dose-dependent manner with a 50% inhibitory concentration (IC50) of 0.104 mM and a 90% inhibitory concentration (IC90) of 1 mM (Fig. 1c), which was further supported by a 640-fold mRNA reduction at 1 mM 6-TG (Fig. 1d), a glycoprotein D reduction by 90% at 3 mM (Fig. 1e), and the significantly reduced viral particles in the culture supernatants (Fig. 1f). 6-TG was effective against infection of five variant strains of HSV-1, including two GCV-resistant mutant strains, HSV-1/blue and HSV-1/153 (Table 1)
We found that HSV-1-infected HCECs at an multiplicity of infection (MOI) of 1 exhibited increased Rac1 activity (;3 fold) by the glutathione S-transferase (GST) pulldown assay (Fig. 5d)

Summary

Introduction

Herpes simplex virus 1 (HSV-1) infects eye corneal tissues leading to herpetic stromal keratitis (HSK), which is one of the leading causes of blindness. We found that 6-thioguanine (6-TG), a once clinically approved medication for child acute myelogenous leukemia, inhibited multiple strains of HSV1 infection in vitro and in vivo. Mechanistic study showed that 6-TG inhibited HSV-1 replication by targeting Rac activity in HSV-1 infected cells, and the Rac is critical in the pathogenesis of HSK. Animal model studies showed that gel-formulated 6-TG topically applied to eyes locally infected with HSV-1 could significantly inhibit HSV-1 replication, alleviate virus-induced HSK pathogenesis, and improve eye conditions. Previous studies have shown that HSV-1 replication relies on Rac1-mediated pathways in keratinocytes, kidney cells, and human umbilical vein endothelial cells [33,34,35]. We wanted to know whether 6-TG could have direct effects on HSV-1 replication by targeting Rac1-mediated pathology

Methods

Findings

Conclusion