抄録
Background: Recent discovery of a small-molecule binding pocket on oncogenic K-Ras with functional significance has prompted several studies that identified potential Ras binders. These include andrographolide (AGP) and its derivative, SRJ23 (3,19-(3-chloro-4-fluorobenzylidene)andrographolide). The latter showed anticancer activity by binding to mutant K-Ras at a specific binding pocket located between two switches of the protein (Hocker et al., 2013, PNAS, 110:10201-10206). The present study aims to investigate the structural relationships between the compounds and K-Ras oncoproteins via in silico simulations, and subsequently to confirm their potential interaction through saturation transfer difference-nuclear magnetic resonance (STD-NMR) study.
Methods: Molecular modelling simulations, including docking and molecular dynamics (MD), were carried out using Glide v6.6 and Desmond v4.1 (Schrodinger, Inc.), respectively to identify the binding affinity of AGP and SRJ23 to wild-type and mutant K-Ras, as well as molecular interactions that lead to their effective biological activity. STD-NMR was carried out using GDP-bound K-RasG12V, to discover the binding nature and tendency of SRJ23 to interact with the oncoprotein, as well as to identify the functional groups involved in the binding events.
Results: Among the K-Ras proteins used to develop molecular docking complexes with AGP and SRJ23, the latter was found to bind strongly and selectively to one of the K-Ras mutants (K-RasG12V, Figure 1A). The compound anchored into the binding pocket via its lactone ring, while its benzylidene ring was found protruding upwards in the groove between switches I and II (Figure 1B). The MD simulation showed the 14-OH group in the lactone ring of SRJ23 was held stably via hydrogen-bond interaction with aspartate-54. Other parts, principally the three-ring system, interacted via hydrophobic contact within the binding site. The STD-NMR assay showed a positive binding of SRJ23 to K-RasG12V, demonstrated by the close proximity of protons (14-OH and three -CH2 moieties of the three-ring system) of SRJ23 to the protein.
Conclusion: SRJ23 binds strongly and interacts stably with K-RasG12V oncoprotein to exhibit its inhibitory activity.
