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HIV-1 Protease Inhibitors from Inverse Design in the Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant VariantsAltman et al. JACS 2008, 130 6099-6113Presented By Swati Jain1Drug ResistanceMutations in drug target selective lower inhibitor affinity maintenance of normal function.Approach drugs for known resistant mutants.Problems potential to introduce new drug resistant mutations.New techniques not induce viable mutations, work with unknown modes of resistance. 2Substrate envelope Hypothesis

Figure taken from : Altman et al. JACS 2008 130 (19) 6099-61133Inverse Inhibitor Design AlgorithmGenerate substrate envelope.Select scaffolds. Choose functional groups.Generate conformational ensembles. Place scaffold in the substrate envelope single and pair-wise energies - DEE/A* - energy ranked compounds.Refine the list - more accurate energy functions.4HIV-1 Protease as target modelHomodimer each subunit made up of 99 amino acids.Well studied proteinAspartic protease: Asp-Thr-Gly active site.

Figure taken from Wikipedia.5Known HIV-1 Substrates and Inhibitor

Figure taken from King et al. Chem bio 11 1333-1338.6Substrate and Maximal Envelope

7Substrate and Maximal Envelope

8Scaffold and functional groupsFunctional GroupsAmprenavir scaffoldCarboxylic acids R1. Primary amines - R2. Sulfonyl chlorides R3 Criterion: < four rotatable bonds. (ignoring the bond to the active group).

Figure taken from : Altman et al. JACS 2008 130 (19) 6099-61139Conformational EnsemblesHydrogen atoms placed at attachment sites for both scaffold and functional groups.Geometry Optimization.Scaffold and Functional Groups: Sampling dihedral angles about each rotatable bond. (every 30 degrees for sp3-sp3, sp2-sp3 and every 45 degrees for sp2-sp2 bond).

10Energy calculationsSubstrate bound protease structureInactivating mutation reversed. Assigned force field parameters.Substrate envelope placed inside the active site.Three components: Van der Waals packing term, screened electrostatic interaction term, Desolvation penalties for both ligand and receptor.

11Grid based energy calculationsReceptor shape and charges fixed.Basis points within the ligand points of cubic grid inside substrate envelope.Van der Waals energies each atom type at each grid point.Electrostatic 1 electron charge at each grid point.Desolvation change in solvation potential for all grid points when one grid point is charged.

12Energy calculations contd Van der Waals energy interpolating energies from grid points.

Electrostatic and desolvation projecting partial charges to grid points.

Figure taken from Wikipedia.13Scoring functionConstant term Binding energy of blunt scaffold + receptor desolvation term.Self energy of functional group Binding energy with receptor + desolvation between functional group and scaffold.Pair wise energies desolvation penalties between two functional groups.Clashes energy infinite.14Scaffold into the EnvelopePlaced the scaffold in the envelope.Scaffold position accepted all atoms within the envelope + required hydrogen bonding + no clashes.For each scaffold placement discrete ensembles of every functional group attached self energies.Pairs of functional groups attached pair wise energies.15DEE/A*Self and pair wise energies sum to the total energy calculated.For each scaffold (backbone) conformation ensemble (rotamers) of functional groups (side-chains) and the self and pair wise energy contribution to the total energy.Used DEE/A* to generate the list of energy ranked conformations.A common list for all scaffold positions.16Hierarchical energy functionsAssumption energies calculated using substrate envelope.Generated list re-evaluated.More sophisticated energy function true molecular surface.Higher Grid resolution.17First Round DesignDesign repeated eight times Tight and loose substrate envelopeDoubly deprotonated and deprotonated protease structure.Rigid and flexible scaffold placement.20 compounds selected based on robustness to parameters.15 synthesized and tested.

18First round Inhibitor Affinities

Figure taken from : Altman et al. JACS 2008 130 (19) 6099-611319Second round designSelection of functional groups based on successful compounds from the first round.Inhibitor bound protease structure used for the design.Only doubly-deprotonated protease structure.Tighter definition of substrate envelope.36 compounds synthesized and tested. 20Second round design results

Figure taken from : Altman et al. JACS 2008 130 (19) 6099-6113Table 2 of paper21

Binding Affinities Drug resistant proteaseinhibitorWTM1M2 M3M4 worst fold lossritonavir0.0553.00.462.8ND55saquinavir0.065901.078ND1385indinavir0.18340.7321ND189nelfinavir0.28153.519ND68lopinavir0.0056.10.0400.90ND1220amprenavir0.100.150.211.40.3414atazanavir0.0460.330.0090.49ND11tipranavir0.0880.0140.0010.032ND0.36darunavir0.0080.0050.0410.0250.334111b422608579ND611c5038066140ND812h332702995ND812j53140130670ND1322Binding Affinities Drug resistant proteaseinhibitorWTM1M2 M3M4 worst fold loss27a0.141.50.0202.00.841427b0.243.00.799.7ND4028a0.0275.90.121.81.221928b0.127.60.452.6ND6329a0.120.990.0641.64.13429b0.0623.50.847.05.311330a0.0360.440.310.570.101630b0.0630.930.496.55.410330d0.0631.10.885.01.37932c0.0140.410.0942.40.2417123Correlation between calculated and observed binding free energies

Figure taken from : Altman et al. JACS 2008 130 (19) 6099-611324Crystal structures of the inhibitorsStructures done four first round, five second round.Scaffold preserved hydrogen bonding network.First round inhibitors mostly inside substrate envelope except one functional group.Second round inhibitors Mostly inside substrate envelope with one exception.25Predicted and Determined structures

Figure taken from : Altman et al. JACS 2008 130 (19) 6099-611326Substrate envelope

Figure taken from : Altman et al. JACS 2008 130 (19) 6099-611327Crystal structures Relation to Resistance profile.

Figure taken from : Altman et al. JACS 2008 130 (19) 6099-611328Testing the algorithm for separating binders and non-binders

Figure taken from: Huggins et al. Proteins 75: 168-186.29Differences from earlier algorithmGeometry Optimization of the Protein structure.Scaffold and side groups - the set of known binders and non binders.Maximal envelopeTorsion angle of the bond attaching functional group to scaffold 10 degrees.Minimization.30Enrichment for binders

Figure taken from: Huggins et al. Proteins 75: 168-186.31Contribution of electrostatic energy

Figure taken from: Huggins et al. Proteins 75: 168-186.32Explicit water model

Figure taken from: Huggins et al. Proteins 75: 168-186.33Issues and ImprovementInhibitors have lower binding energies outside the substrate envelope factors beyond substrate envelope important.Finer Sampling - better results generates too many placements.Scoring functions minimization gives better results MinDEE??.Flexible receptor.Certain functional groups and solubility prediction.34