Computer Aided Drug Design and
Hits identification
La struttura tridimensionale della proteina è stata determinata mediante cristallografia a raggi X o NMR
oppure
è stata ottenuta per homology modelling
De novo drug design BuildingRicerca con gruppi funzionali partendo da un sito ed espandendosi nel sito attivo.
LinkingRicerca con gruppi funzionali partendo da diversi punti del sito attivo e collegandoli successivamente con uno scaffold.
In silico screening
Docking da librerie virtuali o reali
Scoring function
Modification of ligands in situ or design of new ligands
Drug Design
Docking of designed ligands into the binding site (AutoDock, Dock, FlexX…)
Score of the new complexes
Synthesis of new compounds
The Master Equation
G = H - TS
ab
c
dceg
f
H is relatively easy to calculate
• Enthalpy (H) is derived from static models of molecular or bimolecular structure.
• Molecular mechanics force field methods deconvolve H into intramolecular and intermolecular terms from bond stretches, angle bends, torsions, etc., electrostatic interactions and van der Waals (London) forces.
• Many academic and commercial force field programs are available, using similar approaches with essentially comparable results.
S is much harder to calculate
• Entropy = disorder. Computers don’t like disorder!
• Must account for all components of the system, including solvent molecules. (Explicitly?)
• Must add “movement” to the molecule(s) using something like molecular dynamics.
• Entropy itself is not directly (experimentally) measured – calibration of model calculations is less reliable than for enthalpy.
How can we calculate G directly?
• Free Energy Perturbation method (Kollman, Karplus, Beveridge and others)Thermodynamic cycle: Gbind = GI2 - GI1 = Genz – Gsol
(Genz = free energy binding difference between two ligands I1 and I2, Gsol = solvation energy difference between I1 and I2)
Genz and Gsol calculated from extensive molecular dynamics simulations of enzyme/inhibitor systems.
• MC/MD LR (Monte Carlo/Molecular Dynamics with a Linear Response method (Jorgensen et al.)G = [Helec] + [Hvdw] + [SASA]106 to 107 analyzed configurations
Metodi di predizione
• Decomposizione del G° (la teoria dice che è una procedura scorretta)
• Metodi FEP (Free Energy Perturbation) [Kollman, Karplus, Beveridge….]
• MC/MD LR (Monte Carlo/Molecular Dynamics con un metodo Linear Response) [Jorgensen et al.]
• Metodi empirici
A “natural” force field• No preservatives and 9944/100% Hamiltonian
and wave function free!• Biological binding events are not a neat set of
terms specific to hydrogen bonding, acid-base, Coulombic and/or hydrophobic interactions – binding is a concerted process!
• Design a Free Energy force field derived from an experiment that measures the free energy of molecular interactions.
Log Po/w = G° / 2.303 RTThe partition of a compound between water and octanole is a process driven by intermolecular interactions between the solvent and the compound, and by hydrophobic interactions, involving solvation-desolvation. These events are the same that take place in the formation of a
ligand-protein complex.
water octanole
Log PA = Log[Aoct/Awater]
Hydrophobicity
• Measured as Water / Octanol Partition Coefficient (P)
•
• log P > 0 : lipid phase log P < 0 : water phase
Log PA = Log[A]1-octanol
[A]water
Leo (CLOG-P) Method
m
mmn
n
n FjfiLogP11
i = number of occurrences of the fragment constant f of type n.
j = number of occurrences of the factor F of type m.
What is HINT?
Software model based on experimental LogPLogPO/W O/W values for interaction classification and quantitative scoring evaluates enthalpy and entropyenthalpy and entropy
HINT calculates empirical atom-based hydropathic parameters that encode all significant intermolecular and intramolecular non-covalent interactions implicated in drug binding or protein interactions and folding.
The hydrophobic atom constants are calculated using an adaptation of the fragment constant methods of Leo and Rekker.
HINT (Hydropathic HINT (Hydropathic INTeraction)INTeraction)
The “HINT equation”
HINT SCORE = bij = aiSi ajSj Rij Tij + rij
a = hydrophobic atom constantS = solvent accessible surface areaRij = exponential (e-r)Tij = discriminant function for polar-polar interactionsrij = van der Waals term
ai = Log Po/w = - G / 2.303 RT
bij = f (G)
(G. E. Kellogg and D. J. Abraham Eur. J. Med. Chem.2000, 35, 651-661)
Classes of Non-Covalent Interactions
Hydrogen Bonds - A-H…B- 1 – 10 kcal/mole
Coulombic -A+ B-- 0.5 – 5 kcal/mole
Hydrophobic -CHn CHm- 0.5 – 2 kcal/mole
Van der Waals (London) -X Y- < 1
kcal/mole
Note: Any comprehensive method that attempts to model ligandbinding must also consider the energy of solvation and entropiccontributions to the binding process.
Hydropathic Interactions
Coulombic Repulsion
Acid-Base (Hydrogen Bond)
Hydrophobic-Polar (desolvation)
Polar Lewis Base (H-Bond Acceptor)
Acid-Base (Hydrogen Bond)
Coulombic Repulsion
Hydrophobic-Polar (desolvation)
Polar Lewis Acid (H-Bond donor)
Hydrophobic-Polar (desolvation)
Hydrophobic-Polar (desolvation)
Hydrophobic Interaction
Hydrophobic
Polar Lewis Base (H-Bond Acceptor)
Polar Lewis Acid (H-Bond Donor)
Hydrophobic
Negative interactions
Asp-Asp Interaction
Leu-Leu Interaction
Correlazione tra l’energia libera di legame tra proteina e ligandi
determinata mediante HINT e l’energia libera determinata
mediante metodi sperimentali
Model BuildingModel Building• Starting point: protein-ligand complexes for
which 3D structure (PDB files) and experimental binding affinity are determined
• Hydrogen atoms added and minimized, hydrogen bound to polar atoms examined and optimized
Working Working ProcedureProcedure
• Evaluation of the protonation state of ionizable groups on protein and ligand (Computational Titration - Fornabaio et al. J. Med. Chem. 2003, 46, 4487-4500)
• Optimization of water molecules bridging protein and ligand
Hydropathic Analysis for the evaluation Hydropathic Analysis for the evaluation
of all contributions to the ligand-of all contributions to the ligand-protein complex formationprotein complex formation
93 complexes93 complexes formed by 18 different proteins of know 3D structure, R < 3.2R < 3.2 Å Å
General Relationship between Hint Score and General Relationship between Hint Score and G°G°
Hint Score units0 1000 2000 3000 4000 5000 6000
G
° (Kc
al/m
ol)
-20
-16
-12
-8
-4
0
bovine thrombinhuman thrombinhydroxynitrile lyaseadipocyte l.b.p.retinol b.p.bovine trypsin
tryptophan synthasepenicillopepsinsaccharopepsinHIV-1 proteaseothers
G° = -0.0018 HINT score – 3.9041G° = -0.0018 HINT score – 3.9041 R R22 = 0.47 R = 0.68 = 0.47 R = 0.68 se = se = 2.33 K2.33 Kcal/molcal/molCozzini et al., J. Med. Chem. 2002, 45, 2469-2483
73 complexes73 complexes R < 2.5 ÅR < 2.5 Å and at least 3 ligands for each protein
General Relationship between Hint Score and General Relationship between Hint Score and G°G°
Hint Score units0 1000 2000 3000 4000 5000 6000
G
° (Kc
al/m
ol)
-20
-16
-12
-8
-4
0
G° = -0.0024 HINT score – 2.2187 RG° = -0.0024 HINT score – 2.2187 R22 = 0.65 R = 0.81 = 0.65 R = 0.81 se = se = 1.89 K1.89 Kcal/molcal/molKellogg et al. J. Mol. Graph. Model. 2004, in press
Which is the contribution of water Which is the contribution of water molecules bridging ligand and protein to molecules bridging ligand and protein to
the free energy of binding?the free energy of binding?
Active site of HIV-1 Active site of HIV-1 ProteaseProtease
and presence of a and presence of a ligandligand
in the absence
Water molecules in Ligand Binding to Water molecules in Ligand Binding to HIV-1 HIV-1 ProteaseProtease
• is crystallographically detected in the active site• occupies the same position in all complexes•usually forms four hydrogen bonds – two with the protein and two with the ligand•plays a crucial role in molecular recognition
wat301wat301
wat313, 313’, 313bis, 313bis’wat313, 313’, 313bis, 313bis’•not always crystallographically detected •located in a more peripheral area of the active site
w301w301
w313bis’w313bis’
w313’w313’
w313bisw313bis
w313w313
D25 D125
I150 I50
R108 R8
D29
R187R87
D129
The software GRID (P. Goodford, J. Med. Chem. 1985) was used to locate water molecules in HIV-1 protease active site, when they were not present in the crystallographic structures.
D25 D125
I150 I50
R108 R8
D29
R187R87
D129
Hydrophatic Interaction Map: P-L
Hydrophatic Interaction map: P-L + L-H2O
withoutwithout and withwith wat301
The Role of Structural Waters in The Role of Structural Waters in HIV-1 Protease-Ligand Complexes
RR22 = 0.30 R = 0.55 SE = 1.3 = 0.30 R = 0.55 SE = 1.3 Kcal molKcal mol-1-1
RR22 = 0.63 R = 0.80 SE = 1.0 = 0.63 R = 0.80 SE = 1.0 Kcal molKcal mol-1-1
HINT score units2000 3000 4000 5000 6000 7000
G
° (K
cal/m
ol)
-17
-15
-13
-11
-9
-7
with wat301without wat301
The Role of Structural Waters in Ligand Binding to The Role of Structural Waters in Ligand Binding to ProteinsProteins
G
° (K
cal/m
ol)
-17
-15
-13
-11
-9
-7
2000 3000 4000 5000 6000 7000
-17
-15
-13
-11
-9
-7
Hint Score units
G° (
Kca
l/mol
)
-17
-15
-13
-11
-9
-7
SE = SE = 0.8 0.8 kcal/molkcal/molR = 0.84R = 0.84
Hint Score units2000 3000 4000 5000 6000 7000
-17
-15
-13
-11
-9
-7
with wat301-313with wat301-313
with wat301-313-313’-313bis-313bis’with wat301-313-313’-313bis-313bis’
SE = SE = 1.0 1.0 Kcal/molKcal/molR = R =
0.780.78
with wat301-313-313’-313biswith wat301-313-313’-313bis
with wat301-313-313’with wat301-313-313’
SE = SE = 1.0 1.0 Kcal/molKcal/molR = R =
0.780.78
SE = SE = 1.0 1.0 Kcal/molKcal/molR = R =
0.770.77
O
OH
O OP
O O
H
O
O
O OP
O O
H
O
O
O OP
OO
pH 3.5 pH 4.5 pH 5.5
-H
+H
-H
+H
Asp213Asp33
Asp213Asp33
Asp213Asp33
Hint Score units-8000 -6000 -4000 -2000 0 2000 4000
G (K
cal m
ol-1)
-10
-9
-8
-7
-6
-5pH 5.5
pH 4.5
pH 3.5
pH 3.5 pH 4.5 pH 5.5
The role of pH on Ligand BindingThe role of pH on Ligand Binding Penicillopepsin-Phosphonate Ligand Complex
•Binding constants experimentally determined at three different pH values (Bartlett et al., J. Org. Chem. 1990). •Models corresponding to the different protonations of the two catalytic aspartates were built, assuming that addition of one H decrease in one pH unit.
Computational TitrationComputational TitrationDrop protons into the molecular models, one
hydrogen at a time acidification in silico of the environment of the protein-ligand complexes at the binding site
Model ALL possible cases for each “pH” level (corresponding to a defined number of protons into the model)
Calculate the HINT score for each model, averaging the values that correspond to each “pH” level
Plot the mean HINT score values as a function of the number of added protons
The role of pH on Ligand BindingThe role of pH on Ligand Binding
pH3.03.54.04.55.0
G
° (K
cal/m
ol)
-7.0
-6.5
-6.0
-5.5
-5.0
-4.5
-4.0
G° -6.39 -6.36 -6.12 -5.85 -5.25 -5.03 -4.49
pH 3.10 3.40 3.90 4.30 4.40 4.75 4.90
The Role of pH on Ligand Binding:The Role of pH on Ligand Binding:HIV-1 Protease-Peptidic Ligand HIV-1 Protease-Peptidic Ligand
ComplexComplexFor a complex between HIV-1 protease and a peptidic ligand (Glu-Asp-Leu), binding affinities were experimentally determined as a function of pH.
experimental titration experimental titration curvecurve
(J. M. Louis et al., Biochemistry 1998, 37, 2105-2110)
H2N
HN
ONH
OO
O
O
O
OOO
O
O
O
OO
O O_
_
_
__
__
ASP30
ASP29 ASP25
ASP25
H2N
HN
ONH
OO
O
O
O
OOO
O
O
O
OO
O O_
_
_
__
__
ASP30
ASP29 ASP25
ASP25
H+
?… 8 ionizable groups ………. 4374 models to be evaluated
Glu-Asp-Leu bound at HIV-1 protease …
How many protons should be dropped into the models?How many models should be built?How much time does it take?Which is the most favourable ionization state?
All possible ionization states are modeled and scored automatically with the “COMPUTATIONAL TITRATION” procedure
-8000
-6000
-4000
-2000
0
2000
-8 -7 -6 -5 -4 -3 -2 -1 0 1 2Site Charge
HIN
T Sc
ore
-10
-8
-6
-4
-2
0
2
4
6
pH
G (kcal/m
ol)
Model ScoresNormal Ave.Boltzmann Ave.Louis et al.
345
pH of crystallization
Active site of the complex
neuraminidase-DANA (2,3-didehydro-2-deoxy-N-
acetylneuraminic acid)
Conserved interactions :•Three Arg interact with ligand carboxylate•The hydroxyl groups (O8, O9) of the glycerol side chain hydrogen bonded with Glu276•The hydroxyl O4 sits at the entrance of the pocket formed by Asp151, Glu119 (Glu227)
GLU276
ARG292
ARG371
ARG118
O
O
ASP151
O
O
GLU119O
O
HN
H2N NH2
NH
H2N
H2N
HN
NH2
NH2
HO
O
OGLU277
TYR406
-
-
-
-
+
+ +
HO
OH
OH
O
OH
HN
OO
O
-
Computational Titration: Computational Titration: neuraminidase-inhibitor complexesneuraminidase-inhibitor complexes
Protons
0 1 2 3 4 5 6
HIN
T sc
ore
units
3000
3600
4200
4800
Protons 0 1 2 3 4 5 6
HIN
T Sc
ore
units
4000
5000
6000
Computational Titration:Computational Titration: neuraminidase-inhibitor complexesneuraminidase-inhibitor complexes
HO
OH
OH
O
NH2
HN
O-O
O
O
O
OO
ASP151
GLU119O
OGLU276
HO
OH
OH
O
OH
HN
O-O
O
O
O
OO
O
O
ASP151
GLU119
GLU276
G2AR 92R 3GA 71
R 1GA 1 8 G2AR 92R 3GA 71
R 1GA 1 8
The titration curves show a peak HINT score (maximal binding energy) that The titration curves show a peak HINT score (maximal binding energy) that should correspond to the should correspond to the “optimum” pH for binding“optimum” pH for binding..
TOPOGRAPHIC WATER CLASSIFICATION
HIV-1 protease Lipid Binding Proteinp120GAP GTPase-activating domain
Ribonuclease AHsp90 geldanamycin-binding domain
Total number of analyzed water molecules: 817
H-bond ranking
% w
ater
mol
ecul
es
-10
0
10
20
30
40
0-1 1-2 2-3 3-4 4-5
HINT score
% w
ater
mol
ecul
es0
5
10
15
20
25
30
35
-1
00 -
0
0 -
100
100
- 20
0 2
00 -
300
300
- 40
0 4
00 -
500
500
- 60
0 6
00 -
700
700
- 80
0 8
00 -
900
900
- 10
00
mean HINT score PW: 199mean ranking: 1.6
Next:
Analisi delle classi di molecole d’acqua (in cavità, in superficie, in siti attivi,…)
Analisi dell’interazione tra proteina e DNA
In silico screening
Analisi dell’interazione proteina-proteina
