Abstract
RSV is the primary causative agent of lower respiratory tract infections (RTIs) in prematureinfants, 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) |