Abstract
RSV is the primary causative agent of lower respiratory tract infections (RTIs) in premature infants, children below the age of five, elderly and immunocompromised. Reinfections are common and can give rise to chronic respiratory conditions such as asthma, wheezing and bronchiolitis. The current therapy for RSV infections is limited to supportive care and specific antivirals are only administrated for restricted cases such as high-risk infants or immunocompromised patients. Development of drugs targeting host proteins essential for completion of viral lifecycle holds potential as a viable strategy for antiviral therapy. Targeting a host protein, instead of a viral protein, reduces the risk of viral resistance and may be effective against a range of viruses that utilize the same host factor during different stages of their lifecycle.Exportin 1 (XPO1; also known as CRM1) is the sole nuclear exporter for almost 200 proteins involved in several pathways including cell cycle progression, ribogenesis, DNA repair, inflammatory and apoptotic pathways. RSV Matrix (M) protein utilizes XPO1-mediated nuclear export to initiate and coordinate viral assembly and budding in a time-dependent manner.
Inhibition of nuclear export using Leptomycin B (LMB), the prototypical XPO1 inhibitor, resulted in reduced viral replication in vitro, providing proof of principle that inhibition of XPO1 function reduces RSV replication. However, LMB is unsuitable for therapeutic administration.
The overall aim of this research was to characterize the efficacy of specific XPO1 inhibitors against RSV and screen selected plant extracts for possible anti-RSV and anti-XPO1 activity. The efficacy of reversible XPO1 inhibitors called Selective Inhibitors of Nuclear Export (SINE), namely KPT 335, KPT 185 and KPT 301, as antiviral agents against RSV was characterized. KPT 335, also known as Verdinexor, was the most effective among the selected SINE compounds at reducing RSV replication in vitro. Treatment with KPT 335 had the following effects: (1) low cytotoxicity in A549 and Vero cells, (2) increased nuclear localization of XPO1 in treated cells, (3) reduced
amount of XPO1 was present in the treated cells; and (3) induced delayed cell cycle progression within 24 hours of treatment. Treatment with KPT 335 significantly reduced RSV replication in a dose- and time-dependent manner. RSV replication was markedly reduced at 48 hours post infection regardless of duration of treatment with KPT 335. KPT 185 had similar effects on the cells, however, it is unsuitable for therapeutic administration due to a high SI50 value and was found to reduce RSV replication only on continuous treatment. KPT 301 had no antiviral efficacy
against RSV. Treatment with SINE compounds KPT 185 or KPT 335, but not KPT 301, resulted in selective, transient inhibition of XPO1-mediated nuclear export. This led to increased nuclear accumulation of M protein, slowed cell cycle progression and inhibited NFκβ-mediated proinflammatory signalling. No change in viral filament morphology or number of viral inclusion bodies was observed in infected cells treated with SINE compounds, suggesting viral replication, budding and filament formation are not overly affected by SINE treatment. These results suggest treatment with
SINE compounds reduce RSV replication through a combined effect of reduced XPO1, disruption of M nuclear export and delayed cell cycle progression.
The efficacy of selected naturally occurring XPO1 inhibitors against RSV was also characterized.
The cytotoxicity and antiviral efficacy of Valtrate, Acetoxychavicol acetate, Plumbagin,
Piperlongumine, Curcumin, 18-beta-glycrrhetinic acid and Thymoquinone, were determined. Thymoquinone and Curcumin were effective against RSV at low doses and had low cytotoxicity in A549 and Vero cells. Treatment with these compounds also lead to a limited nuclear retention of nuclear export signal (NES)-carrying Rev protein. Treatment with Curcumin had a dose- and time-dependent effect on RSV replication and caused cell cycle arrest in the G0/G1 phase in infected cells.
In contrast to LMB, the reversible inhibition of XPO1 by SINE compounds caused limited/no damage to the cells and effectively reduced RSV replication. Despite being a cytoplasmic virus and M protein being the only known RSV protein to interact with XPO1, disruption of XPO1-mediated nuclear export is a potentially effective therapeutic strategy against RSV. Treatment with SINE compounds had a selective effect on XPO1 and on the downstream pathways regulated by the transporter. In contrast the plant-derived XPO1 inhibitors are likely to have a broad range of
effects on more than one pathway, with XPO1 being one of their targets. This research provides a pipeline of candidates for future drug development.
| Date of Award | 2019 |
|---|---|
| Original language | English |
| Supervisor | Reena GHILDYAL (Supervisor) & Erin Walker (Supervisor) |