Zulkipli, Ninie Nadia
(2022)
Elucidating the effects of mTOR inhibitors from Malaysian natural products on tuberous sclerosis complex cell line.
PhD thesis, Universiti Sains Malaysia.
Abstract
Mammalian target of rapamycin (mTOR) inhibitors are a highly recommended
first-line therapy for tuberous sclerosis complex (TSC)-associated angiomyolipoma.
Everolimus has been approved by the United States Food and Drug Administration
(USFDA) for treatment of TSC-related angiomyolipoma that do not require urgent
surgery. However, everolimus showed only modest efficacy for angiomyolipoma and
has several other limitations. This present study aimed to identify potential mTOR
inhibitor from Malaysian local sources as an alternative to everolimus in treating
angiomyolipoma. The first stage of this present study involved virtual screening and
docking using in silico approach to identify potential mTOR inhibitors from a local
NADI database. AutoDock Vina was applied for virtually screening everolimus and
more than 4000 substances (ligands) from over 360 plants species simultaneously
against FKBP5 and FRB-domain of mTOR. Everolimus acted as positive control and
benchmark for further selection of potential mTOR inhibitors. AutoDock 4.2.6 was
later applied to identify the binding pose of each substance with the lowest docking
score. The second stage of the study involved cell-based bioassays using UMB1949
cell line as TSC model to validate the in silico finding and evaluate the toxicity of the
substances in vitro. The last stage of the study involved evaluation of the effects of substances on genes expression in the mTOR pathway using reverse transcription
quantitative real-time polymerase chain reaction (RT-qPCR) technique. It was discovered asiaticoside and asiatic acid, two bioactive substances from Centella
asiatica have lower and closer docking score to everolimus (– 11.86 kcal/mol) and
have met the selection criteria as potential mTOR inhibitors. IC50 values of asiaticoside
and asiatic acid were 300 μM and 60 μM, respectively. These values were higher than
everolimus (29.5 μM) but they exhibited comparable antiproliferative activities in
vitro. CAB39, PRKCE, RRAGC, and RPS6KA5 were upregulated by all three
substances, everolimus, asiaticoside and asiatic acid. DEPTOR was commonly
downregulated by all three substances. VEGFC (downregulated by asiaticoside) and
IGFBP3 (downregulated by everolimus and asiatic acid) were also selected as genes
of interest due to their reported roles in aiding tumour angiogenesis and apoptosis, and
possible mTOR inhibition. This is the first study to look at identifying possible
therapeutic potentials of asiaticoside and asiatic acid in TSC disease model that targets
mTOR inhibition. These findings, in combination with our in silico findings, provide
a basis for more research into the mechanisms of action, safety, and efficacy of these
substances as mTOR inhibitors.
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