Unraveling the mechanism of CETP inhibition through Molecular Dynamics

simulations: Insights for rational drug discovery

Revathi.SBT12S020

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

Computer Simulations of Liquids Allen and Tilldeseley

-𝜵E=Fi

Bonded

Non-bonded

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 ChartDocking 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

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.

Results

Dynamic Cross Correlation Analysis

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

Anti-correlationPositive -correlation

Grant, Rodrigues, ElSawy, McCammon, Caves, (2006) Bioinformatics 22, 2695-2696

Principal Component Analysis

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.

Inhibitor bound complexes are more dynamic

than the substrate bound

complex

Protein Dynamics from PCA : PC1

Protein Dynamics from PCA : PC2

PCA : Porcupine plotsPC1 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

The N-terminal distal region of both the complexes become predominantly electropositive over time .

The inhibitor bound complex becomes relatively more electropositive.

0 ns 50 ns 100 ns 150 ns

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

Substrate-bound

Torcetrapib-bound

Anacetrapib-bound

Evacetrapib-bound

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

Principal Investigator : Dr.Sanjib SenapatiMembers of the Computational Biophysics Lab, IITM

HPCE Team:Mr. V. RavichandranMrs.P.Gayathri

THANK YOU…