Module 4

Receptor-Ligand Interactions
 What is a Molecule?

Ĥ Ψ = EΨ
Ĥ is the Hamilton operator defining the operations that need to
be performed with the set of wave functions Ψ (psi) of the
electrons of a molecular system.

E is the system’s potential energy

Ψ2 exhibits the electron density. Each Ψ and its corresponding

energy relate to a single electron. They are called “one-electron
orbitals” denoted as 1s, 2s, 2p, etc.
 Diskrete Approximation: Atom
Corey-Pauling-Koltun (CPK) model
• Van der Waals radius
“effective size”
(in Ångstöm; 1 Å = 10-10 m) OH

Johannes Diderik van der Waals

Nobelpreis in Physik 1910

• Atom = “harte Kugel”

F
Cl
Element r/Å
H 1.2
r
F 1.35
O 1.4 O OH
N 1.55
C 1.7 O
S 1.85
Cl 1.8 O
P 1.9
Diskrete Approximation: Bindung
 Van der Waals Radii

Element vdW radius / Å Approximate abundance

symbol in drug molecules / %
H 1.20
C 1.85 37.2
O 1.40 7.6
N 1.54 4.8
P 1.90 0.7
S 1.85 0.6
F 1.35 0.5
Cl 1.81 0.5
 Bond type Bond Energy /
Bond Lengths & length / Å kJ/mol
Dissociation Energies C-C 1.54 348
C=C 1.34 614
C≡C 1.20 839
C-H (sp3-H) 1.11 415
N-N 1.46 170
C-N 1.47 293
SO2-N 1.68 308
C-S 1.82 272
C-O 1.43 358
C=O 1.23 799
C(sp3)-H 1.09 413
N(sp3)-H 1.01 391
O-H 0.96 366
C-F 1.35 485
C-Cl 1.77 328
C-Br 1.94 276
C-I 2.14 240
 Bond Geometry

Bond type Bond angle

109.5°
(sp3)

120°
(sp2)

180°
(sp)
R1 CCN: 122°
H
N CNC: 116°
O R2 peptide
bond: planar
Darstellungen von Aspirin®

8 7 9
HO O
6 10 12
O 1 5 11
OH 2 4 13
3
Worum es geht, am Beispiel der „Aspirin-Story“

Salicin
(aus Weidenrinde, Entzündungshemmer)
Arachidonsäure
HO
COOH
O-β-D-glucopyranosid

COX
COX
COOH COOH
OH O O COOH
O O
OOH
Salicylsäure Acetylsalicylsäure
PGG2
(Aspirin®, Prodrug)
10.10.1897 Erstsynthese
durch Felix Hoffmann (Bayer)
Prostacyclin Thromboxan A2
• inhibiert COX-1 und COX-2 (1991 entdeckt) Pharmakologischer Effekt
Strukturbasierter Entwurf von Wirkstoffen

Ser530

Acetylierung von Ser530

Cokristallstruktur Salicylsäure+COX-1
(PDB: 1pth)
Diskrete Approximation: Oberfläche

• Molecular Surface
• Solvent Accessible Surface (SAS, Conolly, 1985)
• Solvent Excluded Volume

Solvent-accessible surface
(SAS, molecular surface)
vdW surface

Probe
sphere

Lee-Richards
surface
 Wechselwirkungspartner

Rezeptor + Ligand Rezeptor-Ligand-Komplex

• Protein + Wirkstoffmolekül
• Protein + Protein
• RNA + Protein
• DNA + Protein
• RNA + Wirkstoffmolekül
• Lipid + Protein
…
Kristallstruktur der 30S Untereinheit des Ribosoms
(PDB 1giy @ 5.5 Å)
 Protein + „kleines Molekül“

Rezeptor Ligand

O
N O
H2N N HN
OH
N N
O OH
NH2

Kristallstruktur des Komplexes von Methotrexat

und Dihydrofolatreduktase (DHFR) aus Lactobacillus casei

(PDB 3dfr @ 1.7 Å)

 Protein + „großes Molekül“

Komplex zwischen Botulinum Neurotoxin A

und dem Substrat Synaptosomal-Associated Protein 25
(PDB 1xtg @ 2.1 Å)
 Wechselwirkung

Rezeptor + Ligand Rezeptor-Ligand-Komplex

• Wechselwirkungen erfolgen über

Moleküloberflächen
• mit verschiedenen Wechselwirkungstypen

Polare WWs
Lipophile WWs
Aromatische WWs
 Optimale Interaktion

• Sterische Komplementarität (“lock-and-key”)

• Komplementarität von Oberflächeneigenschaften
• Keine abstossenden (repulsive) Wechselwirkungen
• Der Ligand bindet in einer energetisch bevorzugten
Konformation
Der Rezeptor-Liganden Bindungsvorgang
HO

solvated receptor solvated ensemble

binding site of ligand conformations

O O

Enthalpischer Beitrag
solvated receptor-ligand complex
• Brechen und Bilden von H-Brücken
• Bilden lipophiler Kontakte
HO

hydrogen bond Entropischer Beitrag

N
hydrophobic contacts
charge-assisted hydrogen bond
• Freisetzen von Wasser von hydrophoben
Oberflächen zum Medium
• Verlust von Beweglichkeit/Freiheitsgraden
von Rezeptor und Ligand
O O
The thermodynamics of protein-ligand interaction

Activated complex Receptor-ligand

Receptor (transition state) complex k f orward
Ligand
+ P+L PL
k backward

∆G = ∆H − T∆S
Energy coordinate

∆ Ea ∆Ed

∆E

Interaction coordinate
The thermodynamics of protein-ligand interaction

K eq ≡
k forward
=
[PL]
Kd = Ki =
[P] ⋅ [L]
k backward [P] ⋅ [L] [PL]
Equilibrium constant Dissociation constant

 [PL] 
∆G = ∆G° + RT ln 
 [P ] ⋅ [L] 
Equilibrium condition: ∆G = 0 (steady state)

 [PL]   1 
∆G° = − RT ln ( )
 = − RT ln K eq = − RT ln  = RT ln(K i ) = 2.303 ⋅ RT log(K i ).
 [P][L]   Ki 

∆G° = standard free energy change

R = 1.99 cal × mol-1 × K-1 = 8.31 J × mol-1 × K-1
Ki = 10 nM (= 10-8 mol/l)
T = °C + 273.15
∆G° = -47.4 kJ/mol-1.
 Enthalpic Contributions

Noncovalent interaction energies resulting from the

formation and disruption of

• hydrogen-bridges (“hydrogen-bonds”),
• ionic and polar interactions,
• arene-arene (“aromatic”) interactions, and
• dispersive interactions (vdW interactions).
 Entropic Contributions
High-entropy state

HO
O + Lys
H 3N

N
+
Phe

Free solvated ligand Solvated binding pocket

Hydrogen bridge
O (ionic interaction)
H 3N
O

Aromatic
interaction

Solvated ligand-receptor complex

 Entropic Contributions

• The contribution of the hydrophobic effect to

binding is approximately proportional to the size of
the lipophilic surface area shed by the ligand in the
complex and is attributed to 80-200 J/(mol × Å2)

• Bioactive ligand conformations

are within a 5-10 kJ/mol interval to
global minimum conformations in
water
Src kinase

PDB entries 1O4C-K, 104R

 Strong Receptor-Lingand Complexes

Enthalpy-driven Entropy-driven

Tyr43

Tyr33

a) b)

Complex of egg-white avidin Complex of bacterial streptavidin

with biotin (PDB entry 1AVD) and HABA (PDB entry 1SRE)

∆G° = -77 kJ mol-1; ∆G° = -22 kJ mol-1 and

∆H ° = -134 kJ mol-1 ∆H ° = 7 kJ mol-1
 Statistical Thermodynamics

Deduction of macroscopic observations (e.g., free energy /

enthalpy of protein-ligand binding) from molecular principles

Requires a molecular partition function of a system

(conformer ensemble)
= „How molecules are distributed over the available
energy states“

Free energy differences between two states are (almost)

independent of high energy contributions.

The energy differences between the two states can be

quantified from Monte Carlo (MC) or Molecular Dynamics
(MD) simulations.
 Free Energy Perturbation (FEP)

Break down the physical problem of calculating ∆G° of the

formation of a large receptor-ligand complex into several
smaller steps that are feasible to calculate.

HO O HO O HO O

Step 1 Step 2 O Step 3 O

O O

Initial state Intermediate state 1 Intermediate state 2 Final state

λ=0 λ=1

∆G = ∑ ∆Gi
i
 Thermodynamic Cycle

∆G 1
P+L PL
(Protein + Ligand) (Protein-Ligand complex)

∆G2 ∆G4

∆G 3
P+D PD
(Protein + Dummy) (Protein-Dummy complex)

Relative ligand binding energy

∆∆G = ∆G3 − ∆G1 = ∆G4 − ∆G2

• Only the differences between the two ligands count
Comparison of ligand binding energies
 FEP: Thermodynamic Integration

Setup of molecular dynamícs simulations

for bound and free systems

Equilibration of starting systems L

Complex 1
L
Water box
Definition of starting and final systems
∆G2 ∆G4
Definition of λ-transformations and
number of intervals D
Water box D
Complex 2
Equilibration and sampling
for each interval

Summation (integration)
over all intervals ∆∆ == ∆∆G
∆∆G
∆∆
∆∆G G44-- ∆∆G
G22
 The IC50 Value

IC50 = the concentration of competing

ligand which displaces 50% of the
specific binding of a reference ligand.
bound reference ligand

IC 50
Concentration of

Ki =
[Ligand ]
1 + reference
Kd

Cheng-Prussoff

Non-specific binding

IC50
Concentration of test compound
 Screening Methods

Screening plates Screening plates

for HTS „Intelligent LTS“
for LTS

24 well

96 well
HTS Workstation
Wechselwirkungstypen
Nicht-kovalente Wechselwirkungstypen

O H N

hydrogen bonds
O H O Dopt = 2.8 – 3.2 Å
H
O H N

+
O H N

O H
H ionic interactions
Dopt = 2.7 – 3.0 Å
+
N
O H H

hydrophobic interactions
CH3 H3C

+
N
cation-π interaction

-
metal complexation
2+
Zn S
Lipophile Gruppen

O Cl
O S

apolare Heterozyklen
 Die Wasserstoffbrücke
Covalent limit (40 kcal/mol)
H X--H--X
O O [HF2]-
R R
H2N
O H O PO(OH)2
H2N
very strong
Cl3
O H N CH3 Hydrogen-bonds

strong
Hydrogen-bonds
weak
Hydrogen-bonds
NH4+ ···F-
very weak
NH4+ ROO- OH···O=C
Cl3CH···O=CMe2 Hydrogen-bonds
NH···O=C OH···OH C=CH···OH
NH···S C=CH···O
NH4+···Cl- NH4+ ···π NH···NH CH···FC
CH···Cl- CH···S
NMe4+···X- (NO2 )3CH···O
C≡CH···π CH4···π
OH··· π NH···π CH···ClC CH4+···Ar

Electrostatic limit Ph···Ph van der Waals limit

(15 kcal/mol) (0.25 kcal/mol)
 Geometrie von H-Brücken (I)

100°-180°
β

α >150°

2.8 – 3.2 Å

- ∆H ° • gerichtete WW
O-H ••• N (29 kJ × mol-1, 2.88 Å) • definierte Geometrie
O-H ••• O (21 kJ × mol-1, 2.70 Å)
N-H ••• N (13 kJ × mol-1, 3.10 Å)
N-H ••• O (8 kJ × mol-1, 3.04 Å).
 Die Natur als Vorbild

zum Beispiel …
(Trp)
NH
HO OH
O
HN
HO O H
N OH
O P O
O
OH

(Val)

Phosphoramidon

• gefunden in Peptidextrakten aus der Lupine

• inhibiert Metalloproteasen (z.B. Elastase, Thermolysin)
 Starke Wasserstoffbrücke
Ala113
Phosphoramidat
+ Thermolysin O
O O
X
O N P R
H O O-

PDB: 5TMN
Zn2+

R Kd / µM
X = -NH- X = -O- X = -CH2-
OH 0.76 660 1.4
Gly-OH 0.27 230 0.3
Phe-OH 0.08 53 0.07
Ala-OH 0.02 13 0.02
Leu-OH 0.01 9 0.01
 Geometrie von H-Brücken (II)

„Wasserstoffbrücke“ „Ionische Wechselwirkung“

Ligand
Ligand
r
r
α −
ω

Receptor Receptor
 Bindung von Metallionen

Receptor

Zn2+

“Zinc finger”

DNA Bindung
 „Lipophile“ Wechselwirkungen

δ⊕
δ⊕ δ⊕
δ

δ⊕ δ⊕
δ⊕
+

r r r
r
α
ω

Cation-π
π Edge-to-Face Face-to-Face Aliphatic

Aromatische Wechselwirkung
 Multiple Bindungsmoden

Soakingexperimente mit
Trypsin + Guanidiniumbenzoat
Sp
ez
ifit
äts
tas
ch
e

katalytisches Ser195
Böhm et al. (1996)
Bindungsmoden verschiedener Inhibitoren

Überlagerung von 5 dipeptidischen Elastase-Inhibitoren

Böhm et al. (1996)

 Induced Fit

Veränderung der Proteinstruktur durch die Ligandenbindung

stärkere Bindung
geänderte Struktur des Rezeptors
der Ligand bindet nicht in der berechneten theoretisch
„optimalen“ Konformation

Movie:
Bindung des Substrates (purple) an die
ribosomale A-Site ändert die Struktur des
Peptidyl-Transferase-Zentrums
T. Martin Schmeing, Kevin S. Huang, Scott A. Strobel
and Thomas A. Steitz, Nature 438, 520-524 (2005)
 What is a molecule?

HO O 8 7 9

6 10 12
O
1 5 11
OH 2 4 13
3
2D structure Molecular graph

3D conformation 3D conformation Conformer

(experimental, PDB) (experimental, CSC) ensemble
(computed)
 Kraftfelder: Kraftfeldgleichung

Energy = Stretching Energy +

Bending Energy +
Torsion Energy +
Non-Bonded Interaction Energy.

• „mechanistische“ Betrachtung von Molekülen

• physikalisch fundiert

z.B. MM2, CHARMM, OWFEG, AMBER

 Kraftfelder: Streching-Energy

• schätzt die Vibrationsenergie einer Bindung

∑ k (r − r )
2
E stretching = b 0
bonds

Federkonstante Gleichgewichtslänge
(„Steifheit“)
 Kraftfelder: Bending-Energy

• schätzt die Vibrationsenergie eines Bindungswinkels

Ebending = ∑ kθ (θ − θ 0 )
angles
2

Federkonstante Gleichgewichtswinkel
(„Steifheit“)
 Kraftfelder: Torsion-Energy

• schätzt die Torsionsenergie eines Diederwinkels

Etorsions = ∑ A[1 + cos (nτ − Φ )]

torsions

Amplitude Periode Verschiebung

 Kraftfelder: Non-covalent Energy

• schätzt die nichtkovalenten WWs

qi q j  Bij Aij 
E non −bonded = E Coulomb + E vdW = ∑∑ + ∑∑  12 − 6 
i j 4π ε 0 rij i

j  rij rij 

Ladungs WW Dispersions WW
(„Lipophile“ WW)

Demo Benzaldehyd
Conformer Generator: Torsion Angle Distribution

Potential Energy
Relative frequency

0 30 60 90 120 150 180

Torsion angle τ / degrees

 Knowledge-based Conformations

observed distribution calculated potential

H
CH3
N
1 H3C 50
H
H

p(τ) E(τ) /
kJ/mol

0 0
0° τ 180° 0° τ 180°

Torsion angle
p (τ )
E (τ ) ∝ − ln
pref
„Inverse Boltzmann method“
 DOCK (Kuntz)

1. Fülle die Bindetasche mit Kugeln

2. Plaziere Moleküle (3D DB-Suche)

DOCK-Hit

Cysteinprotease-Inhibitor
Optimierung
(Malaria)
 LUDI (Böhm)

• Finde WW-Zentren
HD: blau
HA: rot
Lipo: grün

• Plaziere Fragmente

• Verknüpfe Fragmente

Böhm et al. (1996)

 Docking ist ein Optimierungsproblem

Energy landscape

Local optimum

Global optimum
Barrier to local search

Aufgaben: Random search

1. Docking (Platzieren) Gradientenverfahren
2. Scoring (Bewerten) Stochastische Suche