Abstract
Background: Among all oncogenic events identified in human cancers, RAS oncogenes are the most frequent one comprising over 30% of cancer cases, with the predominantly mutated isoform being KRAS. The mutation is found primarily in pancreatic ductal adenocarcinoma (PDAC, >90%) and colorectal cancers (up to 50%). Our previous analysis using in silico approach showed an andrographolide derivative, SRJ23 (3,19-(3-chloro-4-fluorobenzylidene)andrographolide), could bind to the K-Ras oncoprotein at a specific binding pocket located between two switches of the protein. The binding inhibits mitogen-activated protein kinase (MAPK) cell signalling. Further chemical modifications of SRJ23 yielded eight derivatives (S1- S8) that are anticipated to have improved binding mode. This study aimed to investigate the anticancer activity of SRJ23 and its derivatives through in silico, in vitro, and in vivo approaches.
Methods: MTT assay was used to assess the in vitro growth inhibition of compounds against pancreatic cancer cell lines harbouring wild-type or mutated KRAS (BxPC-3, wild-type KRAS; Capan-2, KRASG12V; MiaPaCa-2, KRASG12C; PANC-1, KRASG12D). Molecular docking was carried out to identify the binding mode and affinity of SRJ23 and derivatives on K-Ras oncoproteins. Immunoblotting was performed to determine the effects of compounds on GTP-loading of K-Ras and MAPK activation. In vivo antitumor activity was evaluated in athymic nude mice carrying PANC-1 tumour xenografts.
Results: SRJ23 inhibited growth of PDAC at relatively low micromolar concentrations and two of its derivatives (S2 and S8) displayed comparable potency. Subsequent docking simulations had identified S2 to bind relatively stronger to the wild-type K-Ras. However, S8 bound selectively to two of the most prevalent K-Ras mutants (K-RasG12D and K-RasG12V) at the same binding site with comparable binding affinities as SRJ23. There was a greater repression in GTP-loading of K-Ras and decreased phosphorylation of MAPK signalling proteins (c-Raf and Erk1/2) in S8-treated PANC-1 cells. A preliminary in vivo study in PANC-1-xenografted nude mice also revealed a greater anti-tumour effect of S8 in comparison with SRJ23.
Conclusion: S8 inhibits aberrant Ras-MAPK signalling and has presented itself as the most promising compound among the SRJ23 derivatives in terms of K-Ras mutant selectivity and anticancer activity.
