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Oleg Alekseev, Jane Azizkhan-Clifford; Targeting host kinases as potential therapeutic targets for the treatment of herpes keratitis. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5990. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Herpes keratitis (HK) is the most common cause of both cornea-derived and infection-associated blindness in the developed world. Despite the availability of potent antivirals, such as acyclovir and famciclovir, a large number of cases are refractory to these agents and develop into permanent corneal damage that necessitates surgical intervention. Importantly, the emergence of acyclovir-resistant strains of herpes simplex virus (HSV) emphasizes the need for the development of novel approaches to combat HK. The purpose of this study is to identify new therapeutic targets against HSV and to assess their antiviral potential in tissue culture experiments, as well as in more sophisticated models of HK. Specifically, we focused on the involvement of ataxia telangiectasia mutated (ATM) and its downstream target Chk2 in facilitating productive HSV infection in corneal epithelium.
We used two human corneal epithelial cell lines - hTCEpi and HCE - as in vitro models of HK. To generate more physiologically-relevant conclusions, we developed an ex vivo model of HK, where we infected intact human and rabbit corneoscleral buttons that were maintained in organ culture. Using corneas obtained from human donors allowed us to extrapolate our tissue culture findings into a highly relevant experimental system. In addition, we are currently utilizing an in vivo mouse model of ocular herpes to further validate our findings. Small molecule inhibitors of ATM (KU-55933, wortmannin, caffeine) and Chk2 (Chk2 inhibitor II), as well as RNAi against ATM and Chk2, were used to inhibit these two kinases. We used plaque assays and quantitative PCR assays to assess the infectious particle production, genome replication, and transcriptional activity of HSV in corneal epithelium.
Small molecule or RNAi-mediated inhibition of ATM or Chk2 greatly suppressed the replication and transcription of the viral genome, as well as its overall infectious particle production. This was observed in the tissue culture models and, importantly, in organotypically explanted human and rabbit corneas. Ongoing experiments address the molecular mechanisms of ATM activation by the virus, as well as the downstream significance of this activation event.
This study identifies two host kinases - ATM and Chk2 - as potential novel therapeutic targets against herpes keratitis.
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