Virtual Screening Discovery of New

Acetylcholinesterase Inhibitors

Mr.Wichitsak sukhano 535150054-3

Advisor : Dr. Chantana Boonyarat
 Contents

• Drug discovery
• Virtual screening in drug discovery
– Types
– Virtual screening process
• Virtual Screening Discovery of New
Acetylcholinesterase Inhibitors
– CHERM Database
Database
 Drug Discovery process

• Most drug molecules inhibit the activity of a specific

protein by blocking its active site
• Examples of proteins targeted by drugs:
– HIV protease (Ritonavir, Viracept,…)
– Dihydrofolate reductase (trimethoprim,methotrexate)

Lead Lead Pre-

Target Clinical Market
IdentificatiOptimizati clinical
discovery Trials Entry
on on test
Discovering Lead Compounds

VS/HTS
?
 Computer aided drug design
• Why does it bind
in this way?
• How can we make
tighter binding,
more specific
compounds?
 Advantages of virtual screening

• “hit” or success rate can be 10-30%,

whereas in HTS it is usually < 0.1%
• avoids the cost of assaying compounds
that do not fit the binding site
• provides a clear structural hypothesis for
ligand binding mode and interactions with
the protein
Virtual Screening

Ligand-based virtual screening

The 3D structure of the biological target is unknown and a set of

geometric rules and/or physical-chemical properties (pharmacophore
model) obtained by QSAR studies are used to screen the database.

Structure-based virtual screening

It involves molecular docking calculations between each molecule to test
and the biological target (usually a protein). To evaluate the affinity a
scoring function is applied. The 3D structure of the target must be known.
Ligand-based virtual screening

3D-QSAR method that search for relationship between the biological activity of a
set of compounds (with specified alignment) and their three-dimensional
physicochemical properties (so-called molecular fields).
Structure-based virtual screening

Docking software
Ligand Receptor

• The complex quality is evaluated by

the score.

Ligand – receptor complex

Automated docking methods
• the scoring (or energy) function
• the strategy used to search for the lowest
score
Common Docking Tools

Ligand flexibility Suitability for

Method Scoring function
sampling large- VS
Force field or
Dock Incremental build High
contact score

FlexX Incremental build Empirical score High

Conformational
Slide Empirical score High
ensembles
Conformational Gaussian score or
Fred High
ensembles empirical scores

Gold Genetic algorithm Empirical score Low

Glide Exhaustive search Empirical score Low

AutoDock Genetic algorithm Force field Low

LigandFit Monte Carlo Empirical score Low

Pseudo-Brownian Mixed force field
ICM sampling local and empirical Low
minimization score
QXP Monte Carlo Force field Low
Molecular
Docking
 Virtual Screening

: Computational or in silico analog of biological screening

ACD
WDI
NCI

X-ray
Crystallography
NMR
homology modeling
Ranking

Score, rank, and/or

filter a HITS
set of structures using
one
or more computational
 Process - Target based VS
In silico library
Starting Database of Compounds Target-protein Add H
receptor, enzyme
ACD, WDI, NCI, inhouse collection Add charges
Check ionization &
tuatomer
Database Preparation Target Preparation

Molecular Docking:Docking tools

- sampling algorithms
- ligand flexibility
- scoring function

Docking Analysis - screening

Rank by Scoring Function

Post Processing Testing Lead optimization

Database Preparations
Filtering
reduce number of compounds, common filtering protocols
• Lipinski’s rule of five ( solubility & permeability )
MW, logP, …...
• Physical filters ( number of rotatable bonds)
• ADMET ( stability & toxicity - some functional groups)
Expanding
increase various forms of each cpds to cover relevant states
(e.g. ionized forms, tautomers, isomers, conformers)
• 3D structure generation --> 3D- co-ordinates
• assign ionization
• all forms of tautomeric states
• enantiomers of chiral centers
• assign partial charges
• geometric isomers
DATABASE PREPARATION
 Filters
•Chemical filters

Substructure filters to eliminate molecules known to have problems

Filters remove structures not wanted in a succession of screening metho

 Pharmacokinetic filters
• Poor absorption or permeation is more
likely when:
– MW > 500
– LogP >5
– More than 5 H-bond donors (sum of OH and
NH groups)
– More than 10 H-bond acceptors (sum of N
and O atoms)
Lipinski rule of 5
 Other finding

70% of drug-like molecules have:

– Between 0 and 2 H-bond donors
– Between 2 and 9 H-bond acceptors
– Between 2 and 8 rotatable bonds
– Between 1 and 4 rings
 Polar surface area

• Amount of molecular surface due to polar

atoms (N and O plus attached hydrogens)
• Especially good for prediction of oral
absorption and brain penetration
• Polar surface are greater than 140 square
Angstroms has been associated with poor
absorption
 Drugs discovery from VS

• Selective COX-2 inhibitor NSAIDs

• Enfuvirtide, a peptide HIV entry
inhibitor
• Zanamivir, an antiviral drug
• Isentress, HIV Integrase inhibitor
Ghosh et al.
Virtual Screening Discovery
of New Acetylcholinesterase
Inhibitors from CERMN
Chemical Library
The 3 D acetylcholinesterase (AChE)
 Acetylcholine (ACh)
• first neurotransmitter
identified (Otto Loewi,
1921)

• important role in movement:

causes muscle contraction

• also found in brain:

important role in attention,
learning & memory
 ACh with Alzheimer’s disease
• The “cholinergic hypothesis”, they
prolong the effects of the endogenously
released neurotransmitter, acetylcholine
(ACh), by inhibiting the enzyme
acetylcholin-esterase, thereby improving
the cognitive abilities of early stage AD
patients.
 AChE: No inhibition
Ca 2
+

Na+
Muscarinic
ACH
ACH Receptor

Acetylcholinesterase
ACH
ACH ACH
Action Potential ACH
ACH
ACH

ACH
ACH
ACH
Choline Acetate

Presynaptic neuron
Postsynaptic target
 AChE: Inhibition by drugs
Ca 2
+

ACH
ACH
Na+
ACH
Muscarinic
ACH
ACH Receptor

Acetylcholinesterase
ACH ACH
ACH ACH
Action Potential ACH
ACH
ACH ACH
ACH ACH
ACH
ACH

Presynaptic neuron
Postsynaptic target
 Types of cholinesterases

• Acetylcholinesterase • Plasma cholinesterase

◆ Located in synapses (Butyrylcholinesterase)
◆ Substrate selectivity: ◆ Located in plasma
• ACH (non-neuronal)
◆ Substrate selectivity:
• ACH
• Succinylcholine
• Local anesthetics
(procaine)
General structure of AChE
 The knowledge of the 3D structure of AChE

• understanding its remarkable

catalytic efficacy
• rational drug design
• developing therapeutic
approaches to OP
intoxication.
 Acetylcholinesterase (AchE)

Zaheer-ul-Haq et al,(2010)

Catalytic triad Peripheral anionic site (PAS)

- Glutamate (Glu327)
- Serine (Ser 200) -Tyrosine (Tyr 70)
- Histidine (His 440) -Trptophan (Trp 279)
 Acetylcholinesterase (AchE)
• Peripheral anionic site (PAS)
• The mouth of the gorge
• Contains two aromatic amino acid
• Tyrosine
• Trptophan
 (d)
(b) acyl pocket

phe 297

phe 295

Anionic site O
N
O
H
N
N (c)
H (c)
O

(a)
(a)
Esteratic site
• H. Dvir et al. / Chemico-Biological Interactions xxx (2010) xxx–xxx
Pheriperal
site

Gorge

Active site
 Donepezil
O O

(S,S)-(-)-bis(10)-hupyridone
HN

H
N
N
H

NH

Inhibit AChE activity

Protect neuron from beta-amyloid toxicity
 Method
• Virtual Screening by Docking.(Gold)
• Pharmacophore-Based Virtual Screening.
(Catalyst)
• In vitro Tests of AChE Biological Activity.
(method of Ellman et al.)
Virtual Screening by Docking
• Target PDB
-1EVE (AChE: Donepezil complex)
-All water molecules were deleted
• Database
- CERMN database
- 2D to 3D by ChemAxon Package
 Docking
• Gold program ; docking conformations
-A genetic algorithm (conformational
spaces&binding mode)
-population size 100
-number of subpopulation 5
-Size of niche 2
-maximum number of genetic applications
100000
-selection pressure 1.1
 Docking
• Migration 5% of time
• Crossover and mutation 95/95
• Fitness function ; GoldScore and
ChemScore (predict affinity)
 In vitro Tests of AChE Biological
Activity
• Ellman method
• AChE ; lyophilized electric eel (enz)

AChE + DTNB+ Test cpds

5 min

ACh iodide (Substrate)

Measure absorbance at 412 nm

Docking result

Gold program In vitro test

6,626 81 24 4

Structure-based screening
Pharmacophore-Based Virtual Screening

3D Pharmacophor PDB
-1EVE (Donepezil)
-1H22 (Hupyridone)
Catalyst ; definition pharmacophor
aligment(superimpose)
- 3D spatial arrangements of chemical 2
molecule
-3D by tolerances sphere
Principal&MaxOmitFeat set 2, 0
Feature misses and complelte misses kept 11
• Superposition error, check superposition,
tolerance factor weights assign set 2
• PP select function of structure
characteristic
Pharmacophore results
7 pharmacophores
Catalyst
Divided into 3 gr In vitro test

6,626 40 29 16 10

10 cpds > 80%

Ligan-based screening
• PP effeiency better SBS (34% success
rate)
• Both screening show 3,5
• Cpd 3 is the best inh. IC50 45+-10 nM
• Confirm with score 61% 27th is the best
• Cpd 5 is IC50 514+-149nM, is 3rd best of
docking score (58%)and 5th best of pp
• Both of them interx cas/pas (close position
donepezil)
SANTOS ET AL.
 Compound
3

Compound
5

Santos et al.
Virtual Screening Discovery of
New Acetylcholinesterase
Inhibitors By ADAM&EVE
 Efficient Method for High-Throughput Virtual
Screening Based on Flexible
Docking

• Virtual Screening tests

- Target :TC AChE ;1ACL
- Databases : ACD (110,000 cpds),
MAYBRIDGE (47,000 cpds)
- Molecular docking : ADAM&EVE
• In vitro test bioactivity : Ellman method

Mizutani and Itai.

 CONVERTER

Remove H2O EVE

Add charge
Add H
Gasteiger
charge

vdW
Electrostatic
Hits criteria
AMBER

Journal of Medicinal Chemistry, 2004, Vol. 47, No. 20

HITs Criteria
 Results

157,000 cpds

114 cpds Docking

Hits
In vitro
35
EVE & MADA

test
cpds
Ranking
1
3
 Binding mode

Compound Compound
1 4
Mizutani and Itai. Journal of Medicinal Chemistry, 2004, Vol. 47, No. 20
 Conclusion

• speed - reduce hit to drug timeline from 15 yrs

from hit identification to approved drug
• cost
• quality of drug candidate for clinical phase
• decrease attrition rate (90%) in clinical phase

**complimentary approach to exp. HTS to increase

success rate