CB5712809, A Novel keap1 Inhibitor upregulates SQSTM1/p62 mediated Nrf2 activation to induce cell death in Colon Cancer cells

Abstract

Background and Aim: Understanding the structural basis of Keap1, a central regulator of the Nrf2 pathway is crucial for cancer regulation. The recent crystallographic elucidation of Keap1's structure provides insights into its functional domains and potential ligand binding sites, paving the way for targeted drug-discovery efforts. This study aims to identify small molecule with high affinity against Keap1 as a modulator of Keap1, SQSTM1/p62, Nrf2 function in colorectal cancer (CRC) cells. Methods A high-throughput virtual screening approach was used to screen the ChemBridge small library against the Keap1 protein. Atomistic Molecular Dynamics (MD) simulations were conducted using GROMACS, along with Gibbs binding free energy estimations. HCT116 and Caco-2 cells were used to determine anti proliferation. Flow cytometry was used to evaluate target inhibition in HCT116 and Caco-2 cells. Results Identified small molecule CBCB5712809, exhibited a stable and avid interactions with key residues of Keap1. Molecular dynamics simulations demonstrated the stability of the protein-ligand complex over a 200ns trajectory. The MM-PBSA analysis indicated a favorable and stable interaction between CBCB5712809 and Keap1, suggesting its potential as a modulator of Keap1 function. CBCB5712809 suppressed the growth of HCT116 and Caco-2 cells with a GI₅₀ values of 40.07 nM and 102.80 nM respectively. Flow cytometry analysis shows that CBCB5712809 arrested the CRC cells in G₂/M phase of cell cycle and downregulated Keap1 levels while upregulating the SQSTM1/p62 and Nrf2 levels. Conclusion Results of this study provides a basis for further experimental validation to develop CBCB5712809 as a Keap1 targeted chemotherapeutic against CRC.