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Molecular InteractionsLecture 2

Dock-umentary

Overview

• Molecular Recognition

• Historical background

• What’s docking?

• Illustrative example: actin-toxin

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Physics of Molecular Recognition

• Van der Waals• Electrostatics – provide specificity• Hydrogen bonds – provide specificity• Hydrophobic effect – provides stability

– Increase in solvent entropy upon burial of non-polar surfaces

• Solute entropy• Internal energies (“strain”)

Thermodynamics

Equilibrium binding constant, Keq; on-rate kon; off-rate koff of the reaction

Relative binding

energies

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Historical background

“. . . the intimate contact between the molecules . . . is possible only with similar geometrical configurations. To use a picture, I would say that the enzyme and the substrate must fit together like a lock and key.”

Lock, dock and barrel

ReceptorEmil Fischer, Ber. Dtsch. Chem. Ges. 1894, 27, 2985.

versus “induced fit” (Koshland)Proc. Natl. Acad. Sci. USA 44:98–104 (1958)

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Dock around the clock

• 1960’s• Protein structures

– Myoglobin: Nature 1958, Kendrew, Phillips, …– Haemoglobin: Nature 1963, Perutz– Lysozyme: Nature 1965, Blake, Phillips, …

• Levinthal. “Molecular model building by computer” Sci Am 1966.

• Hansch: QSAR – Nature 1962.

Dock around the clock

• 1970’s• Protein Data Bank - 1977• Captopril

– early example of structure-based drug design.

• FRODO (later “O”) – Alwyn Jones• Langridge et al. “Real time color graphics

in studies of molecular interaction”. Science 1981.

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Dock around the clock

• 1980’s

• X-plor – X-ray structure refinement; NMR

• Molecular mechanics – CHARMM

• GRID – Goodford. J Med Chem 1985.

• SGI

• Connolly surface

http://www.biohedron.com/msg.pdf

Dock around the clock

• 1990’s• DOCK. Kuntz et al. 1992. • Empirical scoring functions – Bohm

– GOLD, FlexX.

• Relenza– Nature 1993.

• HIV protease inhibitors– Vacca & Condra. Drug Discovery Today 1997.

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Dock around the clock

• 2000’s

• Genomics

• Better homology models

• Virtual high throughput screening

• Better scoring functions / potential energies

Is there a dock-tor in the house?

1A4GInfluenza neuraminidase

ZanamivirRelenza

1LKMHIV proteaseNelfinavirViracept

1XBBAbl kinaseImatinibGleevec

1TBFPDE5SildenafilViagra

1CX2COX-2RofecoxibVioxx

PDBTargetCompoundDrug

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Structure-based drug design

• Requires (crystal) structure for complexes of some analogues with the biomoleculartarget.

• has contributed to the introduction of 50 compounds into clinical trials and to numerous drug approvals

What’s docking?

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Dock, dock. Who’s there?

Docking: the prediction of the structure of receptor-ligand complexes, where the receptor is usually a protein or a protein oligomer and the ligand is either a small molecule or another protein.

Hickory, dickory, dock

1) Add protons, vdWparameters, and partial charges for both target and small molecule.

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The mouse ran up the clock

2) Calculate solvent accessible surface area of target (shown here coloured by element).

The clock struck one

3) Create negative image of surface features surrounding active site using spheres.

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The mouse ran down

4) Calculate energy grid for target.

Each grid point stores vdW score and charge for that area of space.

Hickory, dickory

5) Match ligand atoms to sphere centresand score against grid.

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DOCK

6) Rank best scoring poses (top ten poses from DOCK run shown here).

The Dock Ness Monster

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Actin

• Important part of cytoskeleton• Muscle cells comprise 10%

actin• 2 forms:

– polar, double helical polymer form, filamentous actin (F-actin),

– Monomeric, globular form (G-actin)

• appears in many aspects of molecular oncology

www.sci.sdsu.edu/movies/actin_myosin.html

Structure of the Glob

• Several crystal structures of toxins bound to actin:

• kabiramide C (1QZ5) jaspisamide A (1QZ6) ulapualide A (1S22) swinholide A (1YXQ) reidispongiolide A (2ASM) sphinxolide B (2ASO) reidispongiolide C (2ASP) aplyronine A (1WUA) bistramide A (2FXU)

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Yueng & Paterson. Angew Chem Int Ed 2002, 41, 4632.

• Natural products: a rich source of potential drugs.

• Find important interactions in the hydrophobic binding site of G-actin across all the structural classes for which X-ray data exist

• Aid future development of small molecule binders to actin

Bio-inspired

Motivation

• Pattenden (Chemistry, Nottingham)– Synthetic ulapualide A derivatives

• Shaw (Biomedical Sciences, Nottingham)– Fluorescence actin-binding cell-based assay

• Can we characterize actin-toxin interactions? Can we explain differences in binding affinity? Can we design new ligands?

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The Chemistry

Docking

• widely used & successful in virtual screening for drug design

• computationally cheaper than MD simulations

• large flexible molecules still challenging

• ‘scan’ the actin binding site using flexible docking of smaller substructures (or fragments) of the macrolides

• AutoDockAtomic affinity isosurface for a portion of G-actin, showing potential for carbon (white), nitrogen (blue) and oxygen (red) as derived from atomic affinity grids.

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Substructure docking

• Interactions relevant• Smaller size

– Cheaper; better convergence– Can access regions not accessible to full

macrolide

• Binding affinities too difficult to calculate anyway (e.g. via FEP)

• Semi-quantitative picture

Sanity check

• For each of the ligands, 10 docking runs converged to the same structure: RMSD < 0.5Å

• x-ray coordinates of kabiramide C after superposition of the C-αatoms of actin in 1QZ5 and 1YXQ (RMSD = 0.71 Å)

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Binding pocket

1. the hydrophobic pocket, where most macrolideshave a large hydrophobic ‘anchor’ (except for bistramide A)

2. the hydrophobic ‘cleft’, where a hydrophobic tail is intercalated and which is responsible for the depolymerization effects of the molecules

3. a region at the other end of the cleft, where only bistramide A has been found to bind.

Visualization

• retain atoms from any structure that contribute > 0.6 kcal/mol to docking score via van der Waals interactions and > 0.3 kcal/mol through electrostatic interactions.

• Hydrophobic interactions - cyan spheres

• H-bond acceptor interactions - red spheres

• H-bond donor sites - blue spheres

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Hydrophobic pocket• Kabiramide A (blue) and

reidispongiolide A (yellow) are shown for reference.

• A—hydrophobic interaction at Gly23

• B — hydrophobic interaction at Ser141, Ala144, Pro332, Ser-338

• C— hydrogen-bond acceptor interaction with Arg147

• D hydrogen-bond donor interaction with Glu334

• E — hydrogen-bond donor interaction with Asp25.

Better• Illustrate the potential use of these interaction maps• Modify structure of kabiramide C in silico � a ligand that

bound more ‘‘efficiently’’ (i.e., docking score / heavy atom was larger) than the original structure.

• Do not consider synthetic feasibility, nor entropic penalties

increased the best docking score by3.2 kcal/mol over kabiramide C - increase of 20% and a decrease in Ki by 100-fold; MW reduced from 943 Da748 Da, i.e., by 20%.Parts in red deleted; parts in green added

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Conclusion

• flexible docking of ligand fragments is a valuable tool to rationalize and unify structural data across several ligands

• simple visual confirmation of several observations• extend analysis to potential interaction sites yet to

be exploited.• For actin, focus on a small number of residues:

– hydrophobic interaction between the trisoxazole ring of the kabiramide C and related compounds at Gly-23,

– hydrophobic interaction between the macrolactone ring of reidispongiolide A at Pro-332

Dock-ing in the free world