Abstract
Activation of the Raf/Mek pathway is critical in colorectal cancers (CRC). Several activating mutations have been reported in CRC, where activation of B-Raf by valine to glutamate at position 600 plays a critical role. This study aims to address CRCs with the use of dual inhibitors for B-RafV600E and Mek kinases by using a multifaceted approach. Computational modeling and high throughput virtual screening of large libraries for the target kinases were studied. Caco-2 and HT-29 cells were used to study the effect of the compound on cell proliferation, and flow cytometry was used to analyze target inhibition in these cells by the compound. Atomistic molecular dynamics simulations of the compound predicated C4, a lead candidate to effectively bind B-RAFV600E and Mek proteins. C4 had similar binding stability and improved amino acid residue interactions with these targets compared to known inhibitors viz., GDC0879 (B-RAFV600E) and GDC0973(Mek) kinase. The compound effectively inhibited Caco-2 and HT-29 cell proliferation with GI50 values of 201.3 nM and 332.0 nM, respectively. In both CRC cells tested, C4 inhibited the percentage positive B-RafV600E and phospho Mek 1/2 (S221) populations. C4 has been identified as a novel dual Raf/Mek inhibitor against colorectal carcinoma.
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Abbreviations
- APC:
-
Adenomatous polyposis coli
- CIMP:
-
CpG island methylator phenotype pathway
- CIN:
-
Chromosomal instability
- CRC:
-
Colorectal cancers
- EGFR:
-
Epidermal growth factor receptor
- mCRC:
-
Metastatic colorectal cancer
- MSI:
-
Microsatellite instability
- MTT:
-
(3-(4, 5-Dimethylthiazolyl-2)-2, 5-Diphenyltetrazolium Bromide)
- SPC:
-
Simple point charge
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Acknowledgments
Author expresses his gratitude to SiBIOLEAD, Chennai, Tamil Nadu, India, and SMARTBIO LABS, Chennai, Tamil Nadu, India, for the help rendered in this study.
Funding
The author extends his appreciation to the Deanship of Scientific Research at King Khalid University, Abha, Saudi Arabia, for funding this work through grant number (GRP-218-40).
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Supplementary Figure 1
Molecular Dynamics Simulation of B-RafV600E complexed with standard compound GDC-0879. (a) Ligand to protein Root Mean Square Deviation (RMSD) plot for 100 ns simulation. (b-d) Snapshots of simulation trajectories depicting GDC-0879 interactions with B-RafV600E. Interactions were highlighted in yellow. (PNG 1321 kb)
Supplementary Figure 2
Molecular Dynamics Simulation of Mek kinase complexed with cobimetinib. (a) Ligand to protein Root Mean Square Deviation (RMSD) plot for 100 ns simulation. (b-d) Snapshots of simulation trajectories depicting cobimetinib interactions Mek kinase. Interactions were highlighted in yellow. (PNG 1139 kb)
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Otifi, H.M. In silico high throughput screening and in vitro validation of a novel Raf/Mek dual inhibitor against colorectal carcinoma. Biologia 77, 3555–3564 (2022). https://doi.org/10.1007/s11756-022-01197-2
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DOI: https://doi.org/10.1007/s11756-022-01197-2