docking & designing small molecules within the rosetta code framework
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Multi-Residue Multi Ligand Docking
Tuesday September 18th 2012 DEVELOPMENT OF METHODS FOR DOCKING AND DESIGNINGSMALL MOLECULES WITHIN THE ROSETTA CODE FRAMEWORK A doctoral dissertation defense presented byGORDON HOWARD LEMMON
ROSETTADo NOT explain!1Outline of presentationWhat is structural biology?Protein modeling and ligand dockingIntroduction to Rosetta softwareHIV-1 PR/PI binding affinity predictionRosetta software developmentLigand docking with waters using improved Rosetta ligand docking code22Outline of presentationWhat is structural biology?Protein modeling and ligand dockingIntroduction to Rosetta softwareHIV-1 PR/PI binding affinity predictionRosetta software developmentLigand docking with waters using improved Rosetta ligand docking code33What is structural biology?Proteins
DNAStructural Biology is the study of structure and function of biological molecules such as DNA, RNA, and proteins4
The meilerlab focuses on proteins
There are 1000s of different proteins that all have a unique role to play these include proteins that form muscle, hair, and skin, to proteins that perform chemical reactions, forming and breaking chemical bonds.4How big are proteins?5
Water1.51 HHOAmprenavir~17 72 atomsHIV-1 Protease (PR)~54 3163 atoms1 Angstrom () = 1 ten millionth of a millimeterExplain here that most drugs that you pick up at the pharmacy work by binding to specific proteins.Proteins are very large. How is a molecule this large constructed?5Proteins consist of amino acid chains6
How are molecules as large as proteins created?6Protein sequence determines structure7
This protein has 198 amino acids it is actually two chains of 99 AA eachHow can the sequence determine something as complex as 3-D structure? It has to do with the way that amino acids interact with each other.7Protein structure determines functionHIV-1 protease cleaves poly-protein precursors to form functional proteins8
Peptide chainHIV-1 proteaseSequence determines structure, which determines function.These mature proteins plays a role in the activity of the HIV virus8Proteins are dynamic9
9Outline of presentationWhat is structural biology?Protein modeling and ligand dockingIntroduction to Rosetta softwareHIV-1 PR/PI binding affinity predictionRosetta software developmentLigand docking with waters using improved Rosetta ligand docking code1010What is protein modeling?Prediction of protein structure from Sequence alone (de novo folding)
HIV-1 PRAmino Acid SequenceANPCCSNPCQNRGECMSTGFDQYKCDCTRTGFYGENCTTPEFLTRIKLLLKPTPNTVHYILTHFKGVWNIVNNIPFLRSLIMKYVLTSRSYLIDSPPTYNVHYGYKSWEAFSNLSYYTRALPPVADDCPTPMGVKGNKELPDSKEVLEKVLLRREFIPDPQGSNMMFAFF11
Determining sequence is easy, determining structure is hard. If we can predict structure we can understand function.
11What is protein modeling?Prediction of protein structure from 2.Sequence similarity (Comparative modeling)
HIV-1 PR SequencePQITLWKRPLVTIRIGGQLKEALLDTGADDTVLEEMNLPGRWKPKMIGGIGGFIKVRQYDQIPIEICGHKAIGTVLVGPTPTNVIGRNLLTQIGCTLNFHIV-2 PRHIV-1 PR12
+Using EXPERIMENTAL structures as comparison12What is ligand docking?Prediction of structure of protein/ligand interfacePrediction of ligand binding affinity13
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Structure means the position of the small molecule with respect to the ligand.Predicting binding affinity is more difficult.If we can predict ligand binding affinity, then we can make predictions about how tight a potential drug will bind to its target and how specific that binding will be.13Outline of presentationWhat is structural biology?Protein modeling and ligand dockingIntroduction to Rosetta softwareHIV-1 PR/PI binding affinity predictionRosetta software developmentLigand docking with waters using improved Rosetta ligand docking code1414Rosetta protein modeling consists of sampling and scoring15
Point out that this is 15RosettaLigand docking consists of sampling and scoring16
RosettaLigand docking consists of sampling and scoring17
The lowest scoring model we predict will be closest to the true position that the small molecule will assume.17RosettaLigand docking consists of sampling and scoring18
RosettaLigand score functionKnowledge-based score terms19
Score termDefault weightattractive 0.8repulsive 0.4solvation 0.6dunbrack 0.4pair 0.8hbond_lr_bb 2.0hbond_bb_sc 2.0hbond_sc 2.0
Ive talked about H-bonding but there are many terms and each has a default weight.19Outline of presentationWhat is structural biology?Protein modeling and ligand dockingIntroduction to Rosetta softwareHIV-1 PR/PI binding affinity predictionRosetta software developmentLigand docking with waters using improved Rosetta ligand docking code2020
21HIV-1 PR is flexible
Simmerling 200522HIV-1 PR becomes rigid upon PI binding23
HIV-1 protease mutations
WHO drug resistance mutations in red
24Mutation leads to conformational diversityMutations lead to drug resistance. WHO keeps track of these mutations24FDA approved protease inhibitors (PIs)
Tipranavir
Darunavir
Atazanavir
Lopinavir25Medicine: As HIV-1 PR mutates, a patient being treated with one of these PRs stops responding to treatment. So they are switched to a different PR.25Previous PR/PI G predictions failedCheng (2009)Score FunctionCorrelation N=112Number of non-hydrogen atoms0.172X-Score (HPScore)0.341SYBYL (ChemScore)0.276DS (PMF04)0.183DrugScore (PairSurf)0.225AutoDock0.38
Jenwitheesuk E Samudrala R. (2003)26Experimental vs Predicted HIV-1 PR GExperimental vs Predicted!26Defining G and G
27dG = Gibbs free energy.ddG = Binding affinity. Relative binding affinity w/respect to mutation.27176 experimental PR/PI Gs171 PR template structures
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176 PR/PI Gs sequence but not structure34 sequences10 distinct protease inhibitors171 PR structures represent PR flexibility28RosettaLigand PR/PI Gs predictions290.1 5 PI movementsSide chain and ligand rotamer samplingMinimization of PR side chain and PI torsion anglesMC AcceptMinimize Backbone torsion anglesEnergy filterRandom 5 Translation complete rotation of PI171 PR template structures176 Sequence/PI pairs10 Rosetta relaxed models per input (300,960 models)30,096 Rosetta inputs 1000 RosettaLigand docked models per relaxed model(300,960,000 docked models)Top 10% of models by total score for each Sequence/PI pairTop models by interface score for each Sequence/PI pairRosettaLigand DockingPR/PI Gs prediction workflowx6Reweighting score terms improves HIV-1 PR/PI G predictions Score termDefault weightOptimized weightsGGattractive 0.80.710.31repulsive 0.4-0.010.17solvation 0.60.680.15dunbrack 0.40.290.43pair 0.80.800.80hbond_lr_bb 2.00.850.11hbond_bb_sc 2.00.09-0.20hbond_sc 2.0-0.351.71CORRELATIONS (R)0.160.380.5130
Assuming constant unbound G improves PR/PI G predictionsStandard approachConstant unbound approach31Explain the hypothesis about effect of mutation on flexible vs. rigid structure.31Correlation plotsExperimental on X Predicted on Y Default weights: R=0.1632
Previous PR/PI G predictions failedScore FunctionCorrelation N=112Number of non-hydrogen atoms0.172X-Score::HPScore0.341SYBYL::ChemScore0.276DS::PMF040.183DrugScorePDB::PairSurf0.225AutoDock0.38RosettaLigand0.71
33Experimental vs Predicted HIV-1 PR GExperimental vs Predicted!33Outline of presentationWhat is structural biology?Protein modeling and ligand dockingIntroduction to Rosetta softwareHIV-1 PR/PI binding affinity predictionRosetta software developmentLigand docking with waters using improved Rosetta ligand docking code3434
35
Fragment the Ligand
Search database for fragmentsAssemble rotamer librariesSample from libraries during docking
Flexibility through fragments36Ligand fragment rotamers allow efficient flexibility
37Ligand rotamer docking
38How they were created.38Ligand docking with interface design
A54RL50YC9R DHTDHT: DihydrotestosteroneHisF: imidazole glycerol phosphate synthase
HisFDHTEnlarged prostate glandprostate cancerRosettaLigand prediction39Explain that the ligand moves as well. This is very important!39Fragment based screening can greatly expand sampling spaceCongreve, M. et al. Drug Discov.Today 2003,8, 876-877Traditional ScreeningFragment based screening40The idea is that instead of screening libraries of millions of larger compounds, one could screen libraries of several hundred fragments for several independent fragments, then link these together.40Common drug based FragmentsHartshorn M.J. Murray C.W.et.al. J. Med. Chem. 2005 48 403-413
4141RosettaLigandDesignLibrary of small molecule fragmentsPlace fragments in protein binding site-10-123-7-5Select low energy models for refinementDock ligand with flexible protein side-chains and backbone42RosettaLigandDesignLibrary of small molecule fragmentsPlace fragments in protein binding site-8-15-18-10-12Select low energy models for refinementDock ligand with flexible protein side-chains and backbone43Examples of fragments
Carbon
Oxygen
Nitrogen1 connection
2 connectionsCH2 connections
Ntrp connectionsCore fragment4444Random assembly of fragments
45Rosetta ligand design in action46
Low-res search for starting fragment Refine (dock) starting fragmentGrow small-molecule using fragment libraryRefine (dock) 2-fragment complexGrow small-molecule using fragment libraryRefine (dock) 3-fragment complexAdd Hydrogens to unsatisfied connection pointsProtein binding sites are complex
Dethiobiotin (DTB)Inorganic phosphateMg IonsADP47for example a protein binding pocket can have
47Multiple Ligand docking may capture induced fit effectsSerial Docking
Simultaneous Docking 48Induced-fit means that the protein changes its shape as it interacts with the small molecule.
Enzymes that catalyze chemical reactions, either creating or breaking bonds are good examples.48Rosetta multiple ligand docking
49Outline of presentationWhat is structural biology?Protein modeling and ligand dockingIntroduction to Rosetta softwareHIV-1 PR/PI binding affinity predictionRosetta software developmentLigand docking with waters using improved Rosetta ligand docking code5050
Binding of HIV-1 protease inhibitors involves H2O51Translation of water and PI
52Rotation of water and PI
53RMSD measures accuracy of docked models54
6 Angstrom () RMSD
2 Angstrom () RMSD
6 Angstrom () RMSD
2 Angstrom () RMSD Root mean square deviation
RMSD is an average distance over all pairs of atoms.54Protein-centric waters improve HIV-1 protease placement55
Talk about how important these results are for PI development55Ligand-centric waters improve CSAR inhibitor placementCommunity Structure-Activity Resource299 protein/ligand structures with interface waters56
RMSDs vs Rosetta scores57
RMSD on X axis and Rosetta Interface Score on Y axisWith water we are consistently producing low scoring models below 2 A RMSD57Waters improve docking in non-crowded interfaces58
Interface crowdedness correlates with helpfulness of water docking 59
ConclusionsBinding affinity predictions can be improved byOptimizing Rosetta score term weights Ignoring the unbound stateNew RosettaLigand code allowsMultiple ligand dockingFragment based rotamers for greater flexibilityFragment based design of ligandsDocking with waters helps in spacious binding cavities, hurts in crowded binding cavities
60Professional acknowledgementsMeiler LabJens MeilerKristian Kaufmann Sam DelucaSteven Combs
CommitteeDavid TabbRichard DAquilaBrian BachmannJarrod Smith
Molecular Biophysics Training Grant (NIH)
RosettaCommons
61Personal acknowledgments
Church Friends62Personal acknowledgements
63Personal acknowledgements
64
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