An in-silico analysis of the glycosylation inhibitors Brefeldin A and Tunicamycin C in colorectal cancer; characterization of novel targets

Abstract

Colorectal cancer (CRC), a prevalent malignancy in South Africa, is significantly influenced by posttranslational modifications such as glycosylation. This study investigates the complex interactions between genes, signalling pathways, and cellular processes involved in CRC progression and glycosylation. The glycosylation inhibitors, Tunicamycin and Brefeldin A, are known to hinder colon cancer cell proliferation, migration, and invasion, making them potential therapeutic agents. We used Swiss Target Prediction Software to identify target proteins for both compounds and revealed that Protein Kinase C Alpha (PRKCA), Peroxisome Proliferator- Activated Receptor Gamma (PPARG), and Mitogen-Activated Protein Kinase 1 (MAP2K1) are specific for Brefeldin A, and TK1 and PRKCA for Tunicamycin, respectively. These proteins were selected based on their potential role in the glycosylation process and their role in CRC-related pathways. Further, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis disclosed significantly enriched pathways, including Epstein-Barr virus infection, cellular senescence, and cancer pathways. The 3D-crystallographic structures of PrKC1 (PDB ID 6ar4), TK1 (PDB ID 1w4r), PrCK1 (PDB ID: 6ar4) and MAPK (PDB ID: 3eqc) were retrieved from RCSB Protein Data Bank. The compounds BSP and EGCG were downloaded from PubChem. All non-relevant co-crystallized molecules, including ions, crystallographic water, and others, were removed. Missing residues in the proteins were filled in using the MODELLER algorithm on the UCSF Chimera Graphic User Interface. Molecular docking of Tunicamycin C and Brefeldin A was performed with UCSF Chimera, and the docked conformations were visualised in Maestro and Chimera. The complexes with the top docking scores were selected and prepared for molecular dynamics simulation studies to offer structural and dynamic perspectives on the inhibitory potential of the compounds against the target proteins. The Origin Lab software tool was used to post- analyze the docking conformations. Molecular Dynamics simulation was conducted using Graphics Processing Units version of the Particle Mesh Ewald Molecular Dynamics engine in the AMBER18 suite. Our investigation into the dynamic events leading to the proximal binding of Tunicamycin at the pockets of TK1 and PrKC1 suggested that the binding of Tunicamycin induced a conformational perturbation of the 3D structures of these proteins, resulting in a structural deviation that inhibited their activity. Tunicamycin's time-based dynamics indicated a stable pattern, leading to optimal interaction and maximal stabilization in the hydrophobic pockets of TK1 and PrKC1. Binding energy calculations showed a high-affinity interaction of Tunicamycin with these proteins.

Similarly, the structural investigation revealed that the binding of Brefeldin A to Mitogen- Activated Protein Kinase (MAPK) and Protein Kinase C (PrKC1) inhibited their activity. A detailed analysis of active site residues revealed crucial residues that contributed to the binding stabilization of Brefeldin A. It was noted that the Brefeldin A/MAPK complex produced a binding energy of -22.18±4.50Kcal/mol while the Brefeldin A/PrCK1 complex produced a binding energy of -23.90±5.36Kcal/mol. These findings provide crucial insights into designing novel inhibitors of TK1 and PrKC1, potentially blocking glycosylation progression in cancer treatment. This study underscores the potential for exploiting glycosylation inhibition as a therapeutic strategy against CRC, opening avenues to mitigate cancer progression