Unraveling the mechanism of CETP inhibition through Molecular Dynamics simulations: Insights for rational drug discovery Revathi.S BT12S020

Introduction

Coronary artery diseases and CETP Atherosclerosis - leading cause of mortality in industrialized nations. Recent studies have shown that Cholesteryl Ester Transfer Protein (CETP), which modulates the neutral lipid profile in the body between HDL and LDL, is an effective target. Koivuniemi, A., T. Vuorela, et al. (2012). PLoS Computational Biology 8(1).

Cholesteryl ester transfer protein Boomerang shaped protein molecule with a hydrophobic tunnel. Transfers lipids between High Density Lipoprotein (HDL) and Low Density Lipoprotein (LDL) Inhibiting CETP caused reduction of plaques in the arterial walls of rabbits. Qiu, X., A. Mistry, et al. (2007) Nature structural & molecular biology 14(2): 106-113. Zhang, L., F. Yan, et al. (2012) Nature chemical biology 8(4): 342-349.

Currently available inhibitors of CETP Objective To study the mechanism of CETP inhibition by the 3,5-bis(trifluoromethy)phenyl derivatives

Methodology

Molecular dynamics Simulations Biomolecular simulations – Computational microscope for molecular biology. Molecular Dynamics is based on Newton’s laws of motion -𝜵E=Fi Non-bonded Bonded Computer Simulations of Liquids Allen and Tilldeseley

Why do we need HPCE for MD? System under study: Protein of 7500 atoms protein 522 lipid atoms 81 atoms of inhibitor (variable) 268 Na+ 279 Cl- ions 28000+ atoms from water Total of over 300000 atoms

Software used: NAMD2.9 System scales up to 11ns/day with 256 CPUs in VIRGO

Flow Chart Ligand structure optimization by Gaussian 3.0 Docking of the inhibitors to CETP by AutoDock4 Molecular Dynamics Simulations by NAMD2.9 with CHARMM36 forcefield Visualization and Analysis using VMD1.9.1 and GROMACS

Results RMSD as a function of time reveals that the systems have stabilized. RMSF results show that the N-terminal distal region of the inhibitor bound CETP has higher atomic fluctuations in comparison to the substrate-bound complex.

Dynamic Cross Correlation Analysis Anti-correlation Positive -correlation Substrate bound CETP Torcetrapib bound CETP Anacetrapib bound CETP Evacetrapib bound CETP Inhibitor binding increases the anti-correlation between the binding site and distal regions of CETP Grant, Rodrigues, ElSawy, McCammon, Caves, (2006) Bioinformatics 22, 2695-2696 

Principal Component Analysis Inhibitor bound complexes are more dynamic than the substrate bound complex The above plot reveals that the first 20 Eigen vectors are required to describe 80% of the overall dynamics of the protein. Pronk, S., S. Páll, et al. (2013). Bioinformatics (Oxford, England) 29(7): 845-854.

Protein Dynamics from PCA : PC1

Protein Dynamics from PCA : PC2

PCA : Porcupine plots PC1 PC2 Substrate bound Inhibitor bound Inhibitor bound CETP shows greater extent of twisting in PC2 Humphrey, W., Dalke, A. and Schulten, K., "VMD - Visual Molecular Dynamics", J. Molec. Graphics, 1996, vol. 14, pp. 33-38.

APBS Analysis Substrate-bound 0 ns 50 ns 100 ns 150 ns The N-terminal distal region of both the complexes become predominantly electropositive over time . The inhibitor bound complex becomes relatively more electropositive. Torcetrapib-bound Anacetrapib-bound Evacetrapib-bound Baker NA, Sept D, Joseph S, Holst MJ, McCammon JA. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad. Sci. USA 98, 10037-10041 2001

In conclusion.. Inhibitor binding affects the atomic fluctuations at the distal regions of CETP. Inhibitor binding increases the anti-correlation between the binding site and distal regions of CETP. PCA reveals increased dynamics of the inhibitor bound complexes. Dynamic Cross Correlation Analysis and PCA are indicative of differential twists in the inhibitor-bound systems. This twisting results in the progressive exposure of electropositive residues in the N-terminal distal region. Improved electro-positivity at the HDL –sensing N-terminus supports the existing hypothesis of high affinity of inhibitor bound complexes towards HDL having electronegative surface thereby resulting in the formation of non-productive CETP-HDL complex.* *Clark RW, Ruggeri RB, Cunningham D, Bamberger MJ J Lipid Res 2006;47:537–552

Acknowledgements THANK YOU… Principal Investigator : Dr.Sanjib Senapati Members of the Computational Biophysics Lab, IITM HPCE Team: Mr. V. Ravichandran Mrs.P.Gayathri THANK YOU…