HIV-1 Protease Inhibitors from Inverse Design in the Substrate Envelope Exhibits Subnanomolar Bindin...
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HIV-1 Protease Inhibitors from Inverse Design in the Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant Variants. Altman et al. JACS 2008, 130 6099-6113 Presented By Swati Jain. Drug Resistance.

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HIV-1 Protease Inhibitors from Inverse Design in the Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant Variants

Altman et al. JACS 2008, 130 6099-6113

Presented By Swati Jain


Drug resistance
Drug Resistance Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant Variants

  • Mutations 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.


Substrate envelope hypothesis
Substrate envelope Hypothesis Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant Variants

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


Inverse inhibitor design algorithm
Inverse Inhibitor Design Algorithm Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant Variants

  • Generate 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.


Hiv 1 protease as target model
HIV-1 Protease as target model Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant Variants

  • Homodimer – each subunit made up of 99 amino acids.

  • Well studied protein

  • Aspartic protease:

    Asp-Thr-Gly active site.

Figure taken from Wikipedia.


Known hiv 1 substrates and inhibitor
Known HIV-1 Substrates and Inhibitor Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant Variants

Figure taken from King et al. Chem bio 11 1333-1338.


Substrate and maximal envelope
Substrate and Maximal Envelope Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant Variants


Substrate and maximal envelope1
Substrate and Maximal Envelope Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant Variants


Scaffold and functional groups
Scaffold and functional groups Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant Variants

Functional Groups

Amprenavir scaffold

  • Carboxylic 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) 6099-6113


Conformational ensembles
Conformational Ensembles Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant Variants

  • Hydrogen 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).


Energy calculations
Energy calculations Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant Variants

  • Substrate bound protease structure

  • Inactivating mutation reversed.

  • Assigned force field parameters.

  • Substrate envelope placed inside the active site.

  • Three components: Van der Waal’s packing term, screened electrostatic interaction term, Desolvation penalties for both ligand and receptor.


Grid based energy calculations
Grid based energy calculations Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant Variants

  • Receptor shape and charges fixed.

  • Basis points within the ligand – points of cubic grid inside substrate envelope.

  • Van der Waal’s 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.


Energy calculations contd
Energy calculations contd … Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant Variants

  • Van der Waal’s energy – interpolating energies from grid points.

  • Electrostatic and desolvation – projecting partial charges to grid points.

Figure taken from Wikipedia.


Scoring function
Scoring function Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant Variants

  • Constant 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.


Scaffold into the envelope
Scaffold into the Envelope Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant Variants

  • Placed 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.


Dee a
DEE/A* Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant Variants

  • 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.


Hierarchical energy functions
Hierarchical energy functions Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant Variants

  • Assumption – energies calculated using substrate envelope.

  • Generated list re-evaluated.

  • More sophisticated energy function – true molecular surface.

  • Higher Grid resolution.


First round design
First Round Design Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant Variants

  • Design repeated eight times

    • Tight and loose substrate envelope

    • Doubly deprotonated and deprotonated protease structure.

    • Rigid and flexible scaffold placement.

  • 20 compounds selected based on robustness to parameters.

  • 15 synthesized and tested.


First round inhibitor affinities
First round Inhibitor Affinities Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant Variants

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


Second round design
Second round design Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant Variants

  • Selection 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.


Second round design results
Second round design results Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant Variants

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


Binding affinities drug resistant protease
Binding Affinities – Drug resistant protease Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant Variants


Binding affinities drug resistant protease1
Binding Affinities – Drug resistant protease Substrate Envelope Exhibits Subnanomolar Binding to Drug Resistant Variants


Correlation between calculated and observed binding free energies
Correlation between calculated and observed binding free energies

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


Crystal structures of the inhibitors
Crystal structures of the inhibitors energies

  • Structures 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.


Predicted and determined structures
Predicted and Determined structures energies

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


Substrate envelope
Substrate envelope energies

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


Crystal structures relation to resistance profile
Crystal structures – Relation to Resistance profile. energies

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


Testing the algorithm for separating binders and non binders
Testing the algorithm for separating binders and non-binders energies

Figure taken from: Huggins et al. Proteins 75: 168-186.


Differences from earlier algorithm
Differences from earlier algorithm energies

  • Geometry Optimization of the Protein structure.

  • Scaffold and side groups - the set of known binders and non binders.

  • Maximal envelope

  • Torsion angle of the bond attaching functional group to scaffold – 10 degrees.

  • Minimization.


Enrichment for binders
Enrichment for binders energies

Figure taken from: Huggins et al. Proteins 75: 168-186.


Contribution of electrostatic energy
Contribution of electrostatic energy energies

Figure taken from: Huggins et al. Proteins 75: 168-186.


Explicit water model
Explicit water model energies

Figure taken from: Huggins et al. Proteins 75: 168-186.


Issues and improvement
Issues and Improvement energies

  • Inhibitors 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.


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