Targeting KRAS G12X in Pancreatic Cancer: Discovery of Novel Therapeutic Candidates

Abstract

KRAS is a signalling protein that regulates essential cellular processes including cell survival, proliferation, apoptosis and cell migration. It functions as a binary molecular switch cycling between inactive GDP-bound and active GTP-bound states. Oncogenic mutations in KRAS result in a constitutively active protein, leading to dysregulated downstream signalling. KRAS is the most frequently mutated oncogene in pancreatic ductal adenocarcinoma (PDAC), with mutations occurring in over 90 % of cases. The protein has been considered undruggable due to its surface lacking well defined binding pockets, and its picomolar affinity to GTP, making competitive binding challenging. KRASG12R specifically remains one of the most challenging targets for small-molecule inhibitor development. In this work, we aimed to identify small molecule inhibitors targeting KRASG12R using computational approaches including molecular docking, ShaEP similarity scoring and molecular dynamics simulations. Selected compounds, along with previously identified potential KRASG12R inhibitors, were experimentally validated using ligand-protein interaction studies, including microscale thermophoresis (MST) and differential scanning fluorimetry (DSF). The same experiments were conducted on KRASG12D mutant. Finally, the inhibitory effects of the compounds were evaluated in PDAC cell lines harbouring both KRASG12R and KRASG12D mutations. In total, 64 compounds were purchased and analysed in vitro. Approximately 65 % and 80 % of compounds showed binding in MST binding check for KRASG12R and for KRASG12D, respectively. Additionally, 16 % of compounds exhibited high affinity for KRASG12D. For both mutants, 13 compounds produced significant thermal shifts in DSF experiments, indicating ligand-protein interactions. 4 out of 30 compounds demonstrated measurable effects on PDAC cell viability. These findings highlight the effectiveness of integrating computational and experimental approaches in drug development.