Solving Structure Based Design Problems using Discovery Studio 1.7Building a Flexible Docking Protocol

C. M. (Venkat) VenkatachalamFellow, Life Sciences

Dipesh RisalMarketing, Life Sciences

Overview

• Flexible Docking – elements and challenges• Key Pipeline Pilot Components used in Flexible Docking• Validation of Components• Example workflow for Flexible Docking• Validation of workflow• Work in progress

Flexible Docking (aka ‘Induced Fit’)• Input

– Three dimensional structure of protein

– 2D structure of a ligand

• Output– Generate docked ligand poses

• Challenges– Forcefield, Scoring

– Ligand conformational sampling

– Ligand position and orientational sampling

– Flexibility of Protein• Side chain variations, local and global

• Large movements of protein chain

– Placement of water

• Many of these challenges are addressed in DS 1.7 with well-validated and published algorithms

• The well-validated CHARMm Forcefield and engine underlies all algorithms

Value of Flexible Docking• Experimental evidence shows

that there are variations in protein structure observed when different ligands are bound in it.

• Virtual high-throughput screening generally employs a single receptor structure with a wide variety of ligands. Ignoring protein flexibility can lead to erroneous docking results.

• Protein flexibility may play a vital role in the mechanism (pathway) of ligand docking.

Thymidine Kinase:1kim complex from xray

Variation in Side chains in Estrogen: 1err vs 3ert

residue Δchi1 Δchi2

LEU345 -8.6 19.4

LEU346 -3.1 13.6

THR347 -17.2 0

ARG352 -10.2 -12.07

LEU354 3.1 -12.48

GLU385 -15.6 -14.0

ILE389 -14.2 -11.2

TRP393 6.1 -18.2

ARG394 -15.0 -23.3

MET421 -107.7 47.3

HIS524 128.5 -31.3

LEU525 -69.3 44.5

LEU536 120.2 -94.4

TYR537 179.6 82.0

ASP538 -14.0 -55.0

LEU539 32.4 -10.6

1err

3ert

Δchi = chi(3ert) -chi(1err)

Discovery Studio and Pipeline Pilot

• The Flexible Docking protocol requires a Pipeline Pilot Client• Can be run in Pipeline Pilot or Discovery Studio 1.7

In this presentation…• We introduce 3 Pipeline Pilot components and a Flexible

Docking workflow• ChiRotor

– We show that ChiRotor correctly reconstructs selected side chains with/without ligand present

• ChiFlex– We show that this adequately identifies flexible residues in the

binding site and generates a representative set of protein structures with diverse side chain conformations for docking considerations

• CDOCKER– We show that this successfully docks ligands when correct side

chain conformations are present• Flexible Docking Workflow

– We show this workflow produces correct ligand poses in several protein systems using both native and cross docking.

Key Components used to build a Flexible Docking Workflow

Protein (ChiFlex) Side Chain Conformations

Set of protein structureswith diverse side chainconformations

Proteinw/woutligand

(ChiRotor)Side Chain Builder

Protein structurewith only side chainconformations refined

Protein Ligand+ CDOCKERA set of ligand posesdocked into the rigid protein

ChiRotorV. Z. Spassov, L. Yan, P. K. Flook, “The Dominant Role of Side-chain Backbone Interactions in Structural Realization of Amino-acid Code. ChiRotor: a Side-chain Prediction Algorithm Based on Side-chain Backbone Interactions”, Protein Science 16, 1-13 (2007).

CDOCKERG. Wu, D. H. Robertson, C. L. Brooks III and M. Veith, Detailed Analysis of Grid-Based Molecular Docking: A Case Study of CDOCKER- A CHARMm-Based MD Docking Algorithm, J. Comp. Chem. 24, 1549-1562, 2003.J. A. Erickson, M. Jalaie, D. H. Robertson, R. A. Lewis and M. Vieth, “Lessons in Molecular Recognition: The Effects of Ligand and Protein Flexibility on Molecular Docking Accuracy”, J. Med. Chem., 47 (1), 45 -55, 2004

ChiRotor & ChiFlex Schematic1 2

CDOCKER Schematic

Block Diagram of a Flexible Docking Protocol

3D Structure of Protein

ChiFlex: Side Chain Conformations

n Protein Structuresdiffering in side chainconformations

Start: Loop overn Protein Structures

Stage 1: Dock Ligand to Protein using CDOCKER

→ 1 ligand pose

ChiRotor:Rebuild Side chains

Stage 2: Dock Ligand to Proteinusing CDOCKER

Save Ligand Poses andProtein Structure

Flexible Docking Workflow in Pipeline Pilot

Validation

Questions that Validation should address

• ChiRotor– Does ChiRotor (‘Side Chain Builder’) find the experimentally known

side chain conformations? (It is critical that this produces the correct side chain conformations for the Flexible Docking workflow, since during the CDOCKER step the protein is held rigid with the side chain conformations assigned by ChiRotor.)

• ChiFlex– Does ChiFlex (‘Side Chain Conformations’) identify the flexible

residues in a protein and generate a set of low energy side chain conformations for further considerations?

• CDOCKER– Does CDocker give reasonable ligand dockings if the correct side

chain conformations are present in the receptor?

Does ChiRotor construct the correct side chain conformations if a correct ligand pose is given?

ChiRotor:

ChiRotor: Placement of Side Chains in Thymidine Kinase Structures

ComplexRMSD

(Å)Time

(secs)

1e2k 1.09 176

1e2m 1.85 216

1e2p 1.34 211

1ki2 1.9 215

1ki3 1.35 219

1ki4 1.16 322

1ki6 0.76 345

1ki7 0.79 379

1kim 0.74 270

1qhi 1.48 435

2ki5 -1 1.30 274

2ki5 -2 1.02 343

ChiRotor calculation with ligand present

RMSD to Crystal Structure side chain coordinates

Side chains within 4 angstroms from any ligand atom selected for placement

Computing time for Dell M 70 Pentium 2 GHz single processor

Side Chain Conformations in HUMAN CDK 2 COMPLEXED WITH THE INHIBITOR STAUROSPORINE -ChiRotor without the ligand

1aq1ILE10 0.0651

VAL18 0.2585

LYS33 0.519

VAL64 0.2018

PHE80 0.2069

GLU81 0.1778

PHE82 0.4024

LEU83 1.444

HIS84 0.0814

GLN85 0.4196

ASP86 0.8786

GLN131 2.6595

ASN132 1.9583

LEU134 0.4575

ASP145 1.0151 Protein shown with Ligand

Does ChiFlex (‘Side Chain Conformations’) identify the flexible residues in a protein

and generate a set of low energy side chain conformations for further

considerations?

ChiFlex:

Identification of Flexible Residues

Thymidine Kinase (1kim)His58 Glu83 Ile97 Ile100 Gln125 Tyr132 Arg163 Tyr172 Arg176 Arg222 Glu225Lowest RMSD to Crystal Side Chains ~1.0 angstroms

CDK2 (1aq1)Ile10 Lys33 Phe80 Glu81 Leu83 His84 Gln 85 Asp86 Gln131 Asn132 Leu134 Asp145Lowest RMSD to Crystal Side Chains ~1.8 angstroms

Residues identified by ChiFlex as highly flexible (>2 Å)

Does CDOCKER find the correct ligand pose if the correct protein structure is given?

CDOCKER:

CDOCKER Results: RMSD to 41 Crystal Structures

RMSD

0.00

2.00

4.00

6.00

8.00

10.00

12.00

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

RMSD

1apu (PENICILLOPEPSIN )

1uvs (Thrombin)

1htf (hiv protease)

1pgp (PhosphogluconateDehydrogenase )

Proteins

RM

SD

Same Dataset as Erickson et al. J Med Chem (2004) 47:45-55

Flexible Docking Workflow:Validation Results

Flexible Docking of 1kim ligand into 1kim receptor

Xray Ligand is shown in green.Ligand RMSD to xray = 0.8 angstrom

Flexible Docking of 1ki4 ligand into 1kim receptor

Xray Ligand is shown in green.Ligand RMSD to xray = 1.0 angstrom

Flexible Docking Summary

Ligand RMS to Xray(w/wout receptor flex)

System pdb codes (A, B) ResSel1 aA bB aB bA

Thymidine Kinase 1kim, 1ki4 α 1.2 1.1 1.3/1.1 0.9/3.7

Estrogen Receptor 1err, 3ert β 1.3 1.7 1.4/5.65 1.9/5.2

CDK2 1aq1, 1dm2 α 0.7 0.8 1.3/5.7 1.7/1.6

COX2 1cx2, 3pgh β 1.3 1.7 2.7/6.1 2.4/5.1

Neuraminidase 1nsc, 1a4q α 1.4 1.5 2.1/1.8 2.2/4.2

Thermolysin 1kr6, 1kjo β 1.8 4.4 4.2/4.9 1.6/1.9

HIV-RT 1rth, 1c1c α 2 1.8 2.1/8.8 1.7/3.1

Factor Xa 1ksn, 1xka β 1.9 2.4 1.9/7.0 2.1/8.6

Ligand RMS to XRAYw/wout Receptor Flex

nativedocking

crossdocking

1 ResSel – Residue Selection for ChiRotor and ChiFlexα Residues within 3.5Å from Xray Ligand in both ChiRotor and ChiFlexβ Residues within 3.5Å from Xray Ligand in ChiFlex and additional residues in ChiRotor

Strengths of the Approach

• ChiRotor – ability to reliably predict side chain conformations

• Very flexible interface• Power of modifying the workings by modifying

workflow• Extendable interface – adding LOOPER• Docking engine may be replaced• Consistent, validated force field – CHARMm• A rational approach to Ligand…Protein

interaction problem

Further Work in Progress• Improving Efficiency by

– Intelligent Ligand Placement – Protein structure refinement during docking

• More validation over larger dataset• Fine tuning parameters of the workflow

– Number of Protein side chain conformations– CDOCKER docking parameters

Concluding Remarks

• 3 Pipeline components (ChiFlex, ChiRotor, CDOCKER) have been constructed and validation results presented.

• A Flexible Docking protocol employing the 3 components shows promise in handling protein side chain flexibility in Ligand docking.

• This workflow demonstrates the power and flexibility of the DS platform.

Thanks to …• Accelrys

– R&D• Al Maynard (Structure Based Design) • Eric Yan (CHARMm)• Jürgen Koska (Flexible Docking Workflow)• Lisa Yan (Proteins)• Nan-Jie Deng (Simulation, CHARMm)• Velin Spassov (ChiRotor, ChiFlex)• Paul Flook (Direction)

– Marketing• Sylvia Tara

– Application Scientists• > 25 PhD Scientists world wide participating in validation

• External Collaboration– Charles E. Brooks (CHARMm, CDOCKER)