SHOULD ULD I S I STAY: : PRO ROTON ON TRANS NSFE FER R REVI - - PowerPoint PPT Presentation

ShOUL ULD D i S i STAY Y OR SHOULD ULD I S I STAY: : PRO ROTON ON TRANS NSFE FER R REVI VISITED SITED

Paul Czodrowski SPPEXA 2016 // Software for Exascale Computing

2

Proton transfer?

pH protonation state

0.5 1

pKa value

1 14

amino acid pKa value Arg 13.0 Asp 4.0 Glu 4.4 His 6.3 Lys 10.4 Tyr 9.6

standard pKa values

3

pKa calculations?

size of studied systems speed quantum mechanics empirical implicit models molecular dynamics

G kT pK

a

  303 . 2 1

4

pKa calculations?

pKa Gdiss

experimentally determined computed

AH(aq) A-

(aq)+H+ (aq)

Solvent

Not to forget: We get pKa values for all titratable residues!

G (AH) G (A-)

Protein P-AH P-A-+H+

5

Development of peoe_pb: Our partial charge methodology

Poisson- Boltzmann equation

solvent (e=80) protein (e=20)

Implicit solvent model Partial charges Electrostatic potential

Partial Equalisation of Orbital Electronegativities (PEOE)

distribution of  k A k A k B k

q    

      

 

: orbital electronegativity = 0.5*(IP+EA) = a+b*q+c*q2+d*q3

6

Validation of our partial charge methodology peoe_pb

experimental Gsolv [kcal/mol] calculated Gsolv [kcal/mol] r2 = 0.78 RMSD = 1.57 experimental pKa calculated pKa

eProtein=20

Solvation free energies of small molecules pKa values in proteins

7

Protonation changes detected by ITC

ion bind meas

H n H H     

Trypsin Thrombin

8

Protonation effects for trypsin

1x 1c 1d 1b 4 5

Trypsin Thrombin

9

trypsin/1b vs trypsin/1c

*proton

uptake

ITC: n=+0.90 * FDPB: n=+0.51 ITC: n=±0.0 FDPB: n=±0.0 His57: large pKa shift

10

HIV protease – apo state

1HHP 3HVP

catalytic dyad

Asp25 Asp25‘

pKa1 pKa2 Catalytic dyad Experiment 3.1 – 3.7 4.9 – 6.8 Mono-protonated Calculation 3.8 6.8 Mono-protonated

11

DMP-323 bound to HIVP

1QBS

catalytic dyad

Asp25 Asp25‘

pKa1 pKa2 Catalytic dyad Experiment > 7.2 > 7.2 Doubly protonated Calculation 5.3 10.7 Doubly protonated

DMP-323

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What else can one do with pKa calculations?

13

pKa values & covalent bonds?

Addition Reaction of Model Compounds with Glutathione

http://dx.doi.org/10.1021/jm400822z

taken from:

14

The cysteinome of the kinome

http://dx.doi.org/10.1021/jm101396q taken from:

15

Set-up of the calculations

• Per target kinase, all public PDB structures are used
• The ligand is not considered in the calculation
• Processing/Calculation is done by OpenEye‘s protein_pka

Amino Acid Dictionary Protein PDB PB pKa Protein pKas Ligand

16

Analysis of the calculations

Box plots for all CYS pKa values per protein

pKa

Box plots for all CYS SASA values per protein

SASA

CYS pKa value „out of value“ (i.e. >20) CYS model pKa value

That‘s my NULL model

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EGFR: Covalently attacked CYS-797

Cys.939 Cys.950 Cys.818 Cys.775 Cys.781

3w2o SASA: 28,49 Å2

(w/o non-covalent inhibitor)

3w2p SASA: 38,96 Å2

(w/o covalent Inhibitor)

Cys.797

apo structures

Cys.797

Covalently attacked

Cys.797

pKa

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EGFR: Covalently attacked CYS-797

Cys797 pKa SASA

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EGFR: Covalently attacked CYS-797

Experimental determination of the pKa value Work done at Pfizer

Oral communication at the GordonConference ComputerAided Drug Discovery pKa EGFR.CYS797 = 6.53 +/- 0.05 Site point mutation EGFR/CYS at position 796  pKa = 8.43 Site point mutation EGFR/CYS at position 798  pKa = 8.12

20

Summary

pKa calculations are cool

… because

• They tell you something about the

protonation effect of ligand-protein binding

• The nucleophilicity of the CYS residue

seems to be related to the predicted pKa Why not consider protonation changes in long-scale MD simulations?

21

Acknowledgment

• Gerhard Klebe
• Christoph Sotriffer
• Frank Dullweber
• Ingo Dramburg
• Anthony Nicholls
• Mike Word
• Jose Batista
• Gunther Stahl
• Carl Deutsch
• Christoph Scholz

MERCK