Abrogation of oncogenic k-ras function by andrographolide derivatives via in silico, in vitro, and in vivo approaches
The rat sarcoma (Ras) proteins are small guanosine triphosphatases (GTPases) that act as molecular switches in major signalling pathways involved in cell proliferation, differentiation, and survival, such as mitogenactivated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) cascades...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
2021
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/97712/1/FPSK%28p%29%202021%2015%20-%20IR.1.pdf |
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| Summary: | The rat sarcoma (Ras) proteins are small guanosine triphosphatases
(GTPases) that act as molecular switches in major signalling pathways
involved in cell proliferation, differentiation, and survival, such as mitogenactivated
protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)
cascades. Ras exists in three isoforms – K-Ras, H-Ras, and N-Ras.
Approximately 30% of all human cancers harbour Ras mutations, with the most
frequently mutated isoform being K-Ras, which exclusively appears in
pancreatic ductal adenocarcinoma (PDAC). Mutated K-Ras proteins are
constitutively active with the GTPase activity being compromised. Oncogenic
K-Ras is currently a valuable oncology target and its inhibition represents an
important therapeutic strategy. Recent in silico study has revealed a direct
binding of andrographolide (AGP) and its benzylidene derivatives, SRJ09 and
SRJ23, to K-Ras oncoprotein, which abrogated its function and downstream
MAPK signalling. The present study aims to investigate the potential of AGP
derivatives as anti-Ras therapeutics through in silico, in vitro, and in vivo
approaches. The anticancer potential of SRJ09 and SRJ23 has been welldemonstrated
in the human colon (HCT-116) and prostate (PC-3) cancer cells,
respectively. These two cell lines have been made resistant to the compounds
previously and were used in the present study to examine the altered gene
profile in relation to the expression of regulatory genes involved in the
compounds’ anticancer activity using microarray analysis. Regulatory genes
associated with autophagy and apoptotic processes, such as ATG12 and
HMOX1, as well as MAPK and PI3K pathways, such as FGF19 and SPRY2
that play major roles in promoting cell growth and survival, were found to be
altered. New benzylidene derivatives have been previously synthesised using
SRJ09 and SRJ23 as parent compounds, yielding SRS compounds. In the
present investigation, the most druggable binding pocket on K-Ras mutants
namely p2 was revealed through in silico simulations. SRJ23 and SRS157
were found to bind via intermolecular hydrogen bonding to this pocket. The anti-PDAC activity of selected AGP derivatives (SRJ23, SRJ09, SRS07, and
SRS157) and their mechanisms of action were elucidated in vitro. SRJ23 and
SRS157 were shown to perform differently particularly in terms of activity on
Erk, a crucial signalling protein in the K-Ras-associated MAPK cascade. Its
activation was unanticipatedly enhanced by SRJ23 and significantly
suppressed by SRS157 upon 24-h treatment of the compounds. SRS07
presented as a superior anti-PDAC agent by promoting oxidative stress,
possibly through enhancement of Akt activation in the K-Ras-mediated PI3K
pathway. A simple pharmacokinetic study performed in BALB/c mice at a single
dose of 100 mg/kg SRJ23 revealed that the compound achieved a maximum
plasma concentration of 18.8 μM after 30 min of administration, with long halflife
(4.28 h) and mean residence time (6.30 h). Subsequent in vivo antitumour
study reported that 100 mg/kg SRS157 delayed the doubling of tumour growth
in the PDAC-xenograft nude mouse model more effectively than SRJ23 at the
same dose. In conclusion, the outcomes of the present study provide a strong
indication of the potential of AGP derivatives, which specifically target the
oncogenic K-Ras and abrogate its function, as promising clinical antipancreatic
cancer candidates. |
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