Discovery, optimization and biological activity evaluation of genipin derivatives as potential KRAS G12D inhibitors.

A series of genipin derivatives were designed and synthesized as potential inhibitors targeted KRAS G12D mutation. The majority of these compounds demonstrated potential antiproliferative effects against KRAS G12D mutant tumor cells (CT26 and A427). Notably, seven compounds exhibited the anticancer effects with IC50 values ranging from 7.06 to 9.21 µM in CT26 (KRASG12D) and A427 (KRASG12D) cells and effectively suppressed the colony formation of CT26 cells. One representative compound SK12 was selected for further investigation into biological activity and action mechanisms. SK12 markedly induced apoptosis in CT26 cells in a concentration-dependent manner. Moreover, SK12 elevated the levels of reactive oxygen species (ROS) in tumor cells and exhibited a modulatory effect on the KRAS signaling pathway, thereby inhibiting the activation of downstream phosphorylated proteins. The binding affinity of SK12 to KRAS G12D protein was further confirmed by the surface plasmon resonance (SPR) assay with a binding KD of 157 µM. SK12 also exhibited notable anticancer efficacy in a nude mice tumor model. The relative tumor proliferation rate (T/C) of the experimental group (50 mg/kg) was 31.04 % (P < 0.05), while maintaining a commendable safety profile.

A novel Imidazo[1,2-a]pyridine derivative modulates active KRASG12D through off-like conformational shifts in switch-I and switch-II regions, mimicking inactive KRASG12D.

KRASG12D are the most prevalent oncogenic mutations and a promising target for solid tumor therapies. However, its inhibition exhibits tremendous challenge due to the necessity of high binding affinity to obviate the need for covalent binders. Here we report the evidence of a novel class of Imidazo[1,2-a]pyridine derivative as potentially significant novel inhibitors of KRASG12D, discovered through extensive ligand-based screening against 2-[(2R)-piperidin-2-yl]-1H-indole, an important scaffold for KRASG12D inhibition via switch-I/II (S-I/II) pocket. The proposed compounds exhibited similar binding affinities and overlapped pose configurations to 2-[(2R)-piperidin-2-yl]-1H-indole, serving as a reliable starting point for drug discovery. Comparative free energy profiles demonstrated that C4 [2-methyl-3-((5-phenyl-1H-1,2,4-triazol-3-yl)methyl)imidazo[1,2-a]pyridine] effectively shifted the protein to a stable low-energy conformation via a prominent transition state. The conformational changes across the transition revealed the conformational shift of switch-I and II to a previously known off-like conformation of inactive KRASG12D with rmsd of 0.91 Å. These conformations were even more prominent than the privileged scaffold 2-[(2R)-piperidin-2-yl]-1H-indole. The representative structure overlay of C4 and another X-ray crystallography solved BI-2852 bound inactive KRASG12D revealed that Switch-I and II exhibited off-like conformations. The cumulative variance across the first eigenvalue that accounted for 57 % of the collective variance validated this on-to-off transition. In addition, the relative interaction of C4 binding showed consistent patterns with BI-2852. Taken together, our results support the inhibitory activity of [2-methyl-3-((5-phenyl-1H-1,2,4-triazol-3-yl)methyl)imidazo[1,2-a]pyridine] by shifting active KRASG12D to an inactive conformation.