Targets looking for drugs:

a versatile method for the development of

structure-based pharmacophoric models

Prof. Maurizio Botta, Ph.D.

University of Siena
 Virtual Screening
Application of computational filters to screen a large library of compounds
in order to obtain quickly and rationally a restricted number of compounds to
be submitted to biological evaluation
Virtual Library Design
Synthesis

VL
Virtual Screening
(pharmacophore,
docking, QSAR etc.)
M
ay Selection of few
br compounds
id Biological
n k ge
evaluation
Ba DB
e m
Ch
sI nEx Purchase
A Hit identification
Available compounds
 What Is A Pharmacophore?

“A pharmacophore is the ensemble of steric

and electronic features that is necessary to
ensure the optimal supramolecular
interactions with a specific biological target and
to trigger (or block) its biological response”

C.-G. Wermuth et al., Pure Appl. Chem. 1998, 70:1129-1143

 How To Build Pharmacophore Models?

There are two basic approaches for

pharmacophoric model generation, which
depend on the available information about the
3D structure of the target under study.
unknown

known 3D structure
of the target Ligand-based
Pharmacophores (DISCO,
Catalyst, GASP)

Structure-based
pharmacophores
(LigandScout, MOE)
 Receptor-Based Pharmacophores

In view of the rapidly growing number of protein

structures that are now available, receptor-based
pharmacophore generation methods are becoming
more and more used.

Almost all of these procedures require the

knowledge of the 3D structure of the ligand-
protein complex and they convert interaction
pattern into pharmacophore point location.
(G. Wolber et al. J. Chem. Inf. Model. 2005, 45, 160-169.)
 Receptor-Based Pharmacophores

These methods have some limitations:

• they can not be applied when no compounds
targeting the binding site of interest is known
• one pharmacophore model accounts for one
binding mode.
 Receptor-Based Pharmacophores

The generation of receptor-based pharmacophores

starting from the knowledge of the sole target has
received little attention and only few applications
have been reported so far.

In this context, we have applied a versatile

computational procedure able to generate receptor-
based pharmacophoric models starting from the
analysis of the sole protein target.
 Receptor-Based Pharmacophores

The Approach Described Here:

• Does not require the knowledge of the 3D structure of the
ligand-protein complex.

• Allows the generation of structure-based pharmacophoric

models also for receptor sites which have never been
targeted before and for which no ligands are known.

• Could be applied together with other techniques such as

docking studies, molecular dynamic simulations and
conformational analysis.

• Could be integrated in a virtual screening procedure to

improve the drug discovery process.
 Method
I step: Calculation of the
MIFs (Molecular Interaction
Fields) for the binding site
(GRID program). The software
initially builds a grid over the
region of interest, then
computes at each grid point the
interaction energies between
the protein and several probes.

At least three different probes should be used:

a hydrophobic probe (DRY, hydrophobic probe; C3, methyl group;
C1=, aromatic carbon);
a hydrogen bond acceptor (HBA) probe (O, that is a sp2 carbonyl oxygen);
a hydrogen bond donor (HBD) probe (N1, a neutral flat NH group).
 Method
II step: The points of minimum of MIFs were calculated, thus
identifying the regions of most favorable interaction between each
probe and the protein.

The files corresponding to

each MIF were processed by
means of the Minim and
Filmap programs ( GRID
package), collecting all points
within a certain energy value.
A threshold of 0 Kcal/mol has
been used to exclude points
with a positive energy of
interaction.
 Method
III step. Number of points of minimum is
generally too high to allow the construction of
useful pharmacophoric models, a selection
of the most suitable points is necessary.

The criteria for the selection are:

(1) The interaction energy: for each probe,

the points at lower interaction energy should
be preferred.

(2) The position within the binding site:

points located within cavities are more suited
than points exposed on the surface.
 Method
(3) The distance from other minima:

- Too close, could not be simultaneously

mapped by the ligand moieties (minimum
distance between points 1 Å)

- Too distant, could force the selection of only

large ligands (maximum distance 15 Å).

(4) The presence (close to the point of

minimum of N1 and O probes) of a protein
functional group able to act as HBA or HBD,
respectively.
 Method
IV step. The coordinates of the minima were converted on
pharmacophoric features:
- O probe => HBA features
- N1 probe => HBD features
- hydrophobic probes => hydrophobic features
HBD and HBA features were directed toward the atoms of the protein
which act as HB acceptors or donors, respectively (Catalyst 4.10.)
Method

V step. Excluded volume

spheres are added to the
pharmacophore to mimic the
boundary of the active site. They
represent regions that can not be
occupied by any ligand portion.

The use of excluded volumes

allows to codify information about
the shape and the steric
hindrance of the binding site
itself into the pharmacophore
 Outline

• Structure-based virtual screening for the identification

of potential inhibitors of Mycobacterium tuberculosis
thioredoxin reductase.

• Molecular modeling, synthesis and biological studies

of novel HIV-1 integrase inhibitors.

• A target for new antiviral drugs: Inhibition of the

Reverse Transcriptase Dimerization.
 Outline

• Structure-based virtual screening for the identification

of potential inhibitors of Mycobacterium tuberculosis
thioredoxin reductase.

• Molecular modeling, synthesis and biological studies

of novel HIV-1 integrase inhibitors.

• A target for new antiviral drugs: Inhibition of the

Reverse Transcriptase Dimerization.
 The Target: Thioredoxin Reductase
• Thioredoxin reductase (TrxR) is part of the thioredoxin system, which is
responsible for maintaining reducing conditions inside the cell.

• TrxR catalyzes the transfer of electrons between NADPH and thioredoxin,

promoting catalysis via FAD and a redox-active disulfide.
SH S
NADPH FADox
SH S Reductive
cellular
processes
S SH
+ FADred
NADP SH
S

Thioredoxin Reductase Thioredoxin

• In Mycobacterium tuberculosis the glutathione system is absent, thus the

thioredoxin system plays a crucial role for the pathogen’s resistence to the
oxidative stress exerted by the innate immune response.

• No inhibitors of M.tuberculosis TrxR have been reported so far.

 The “Ball-And-Socket” Motion

NADPH
domain
The proper functioning
Cys138

of TrxR involves the Trx

switching between two Cys138

different conformations. FAD

FAD
The twisting of the NADPH
domains relative to one NADP+

another resembles the

twisting of the ball
within the socket.

FAD
FO domain FR
conformation conformation

• FO conformation: the redox-active disulfide (Cys135-Cys138) is buried.

• FR conformation: the active site disulfide is exposed and can interact
with thioredoxin (Trx).
 Available Crystallographic Data

• FO conformation of M. tuberculosis TrxR (PDB entry: 2a87)

• FO and FR conformation of E. coli TrxR (PDB entry: 1tde and 1f6m)

• Sequence identity M. tuberculosis / E. coli TrxR = 45%

• Despite the moderate sequence identity, “the overall structure of M.

tuberculosis TrxR is quite similar to that of E. coli TrxR”
(M. Akif et al. Acta Crystallogr. D Biol. Crystallogr. 2005, 61, 1603-11).
 Two Different Strategies
Aim of our work
Elaborating a structure-based virtual screening protocol to identify
compounds able to inhibit the TrxR activity by:
1) Targeting the TrxR/Trx interaction
2) Targeting the NADPH binding site

NADPH
domain Cys138

Trx
Cys138
FAD
FAD
NADPH
NADP+

FO FAD FR
conformation domain conformation
 1st Approach: Targeting The TrxR/Trx Interaction

Aim of our work NADPH

domain Cys138

Elaborating a structure-based
Trx
virtual screening protocol to Cys138

identify compounds targeting FAD

FAD

the exposed active site of TrxR NADPH

NADP+
and able to inhibit its activity by
affecting the TrxR/Trx
interaction, possibly alkylating
the active site Cys138. FO FAD FR
conformation domain conformation

To reach our goal, it was necessary to model the FR

conformation of M. tuberculosis TrxR, in order to bring the
active site disulfide to the surface of the enzyme.
 Computational protocol

• Homology modeling of the FR conformation of M. tuberculosis TrxR

using the FR conformation of E. coli TrxR as template.

• Creation of the Michael acceptor (MA) pharmacophoric feature.

• Binding site analysis by means of GRID software and identification

of points of minimum in GRID molecular interaction fields for probes
DRY, C3, N1 and O.

• Design of receptor-based pharmacophoric models based on GRID

points of minimum and also containing the MA feature.

• Elaboration of a virtual screening procedure based on

pharmacophoric models and docking studies.
 Definition of the Michael acceptor feature
O O

N
• Compounds reported to
undergo Michael NO2 O
addition by cysteine
residues were collected N
from literature.

• Fragments extracted
NO2
MA CN
from such compounds
were used to create a
new pharmacophoric
O
feature, called MA O O
S N
(Michael Acceptor). [C,O,N]
N

Fragments (together with corresponding centroids) used

for the assembly of the new pharmacophoric feature.
Receptor-based pharmacophoric model

Pharmacophoric
GRID probes: features:

● Hydrophobic
DRY (DRY/Methyl)
● H-bond
Methyl acceptor (Carbonyl oxygen)
● H-bond
Carbonyldonor (Flat NH)
oxygen
● Michael
Flat NH acceptor
● Excluded volume
 Virtual screening procedure

Asinex Database

____
• Parmacophoric search ___________________________

____
• Rotatable bonds ≤ 10 ____________________________

____
• Doking energy __________________________________

____
• Analysis of complexes ____________________________

10 compounds selected
 Ezymatic assays

% inhibition

50
45
40
35
30
50 µM
25
100 µM
20
15
10
5
0

10
1

9
ol
TR

TR

TR

TR

TR

TR

TR

TR

TR
tr

TR
on
C
 2nd Approach: Targeting The NADPH Binding Site

NADPH
Aim of our work domain Cys138

Elaborating a structure-based Trx

Cys138
virtual screening protocol to FAD
FAD
identify compounds able to NADPH
NADP+
inhibit the activity of TrxR by
competing with NADPH.

FO FAD FR
conformation domain conformation

For this kind of approach, it was possible to use the available

crystallographic structure of the FO conformation of M. tuberculosis
TrxR, since in this case the binding site of interest was exposed.
 Computational Protocol

• Analysis of NADPH binding site by means of GRID software and

identification of points of minimum in GRID molecular interaction
fields for probes DRY, C3, N1 and O.

• Design of receptor-based pharmacophoric models based on GRID

points of minimum.

• Elaboration of a virtual screening procedure based on

pharmacophoric models and docking studies.
Receptor-Based Pharmacophoric Model

GRID probes: features:

Pharmacophoric

● Hydrophobic
DRY
Methyl (Methyl)
● H-bond
Methyl acceptor
Carbonyl oxygen (Carbonyl oxygen)
● H-bond
Carbonyl
Flat NH donor
oxygen
(Flat NH)
● Excluded
Flat NH volume
 Virtual Screening Procedure

Asinex Database

• Parmacophoric search ___________________________

_____

• Rotatable bonds ≤ 10 ____________________________

_____

• Docking energy _______________________________

_____

• Analysis of complexes ___________________________

_____

9 compounds selected
 Virtual Screening Procedure

The structure-based pharmacophoric models were applied as

first filter to screen the Asinex Database and led to the selection of
22290 compounds. Next, the number of rotatable bonds and the
Best Fit value were used to remove chemicals either too flexible
or poorly fitting the pharmacophore models, respectively.
 Virtual Screening Procedure

The resulting 9156 compounds were subjected to docking (software

GOLD) in two consecutive runs to optimize the balance between
the quality of docking and the calculations time:

1st. Search efficiency parameter set to 50% to speed up the process.

2st. Search efficiency was set to 100% on the top 1000 scored hits.
 Virtual Screening Procedure

47 compounds for which the binding mode is in agreement with

the pharmacophoric features, were chosen.
In fact, both pharmacophore and docking are aimed at
hypothesizing the bioactive conformation of ligands, so compounds
for which a consensus between the two methods occurs should be
preferred to those for which do not occur.
 Virtual Screening Procedure

9 molecules was finally selected considering:

• predicted binding energy
• different structural classes
 Enzymatic Assays

% inhibition

100
90
80
70 TR12, IC50 = 16 µM
60
50 µM
50
100 µM
40
30
20
10
0
11

12

13

14

15

16

17

18

19
ol
tr

TR

TR

TR

TR

TR

TR

TR

TR

TR
on
C

Assay conditions: Compound: 50/100 µM

NADPH: 225 µM
 Active Compound

O
O
HN
O
O N O O
H
O

a) Alignment of the active compound

(orange) on pharmacophore.

b) Binding mode of the active

compound within the NADPH
binding site of MTB TrxR.

There is a good agreement between pharmacophore mapping

and docking results, the main difference being the location of the
methoxy substituent on the phenyl ring which is not predicted to
be involved in any interaction with the binding pocket.
 Conclusions

• Structure-based virtual screening procedures for the search of

inhibitors of M. tuberculosis TrxR resulted in the identification of
TR1 and TR12 as hit compounds.

• The candidate compounds inhibited the TrxR activity through two

distinct putative mechanisms of action.

• The biological activities of TR1 and TR12 make them worth of

further development (already in progress).
 Outline

• Structure-based virtual screening for the identification

of potential inhibitors of Mycobacterium tuberculosis
thioredoxin reductase.

• Molecular modeling, synthesis

and biological studies of
novel HIV-1 integrase inhibitors.

• A target for new antiviral drugs: Inhibition of the

Reverse Transcriptase Dimerization.
 Anti-HIV-1 Therapy

AIDS (Acquired Immune

Deficiency Syndrome) is
an infection disease
caused by a virus
known as the Human
Immunodeficiency Virus
(HIV).

Highly active antiretroviral therapy

• Nucleoside analog (RT inhibitors)

• Non-nucleoside analog (RT inhibitors)
• Protease inhibitor drugs
• Entry inhibitors
 Role Of Integrase
The Integrase catalyses two temporally and spatially separated
reactions known as 3’-processing and strand transfer.
The 3’-processing occurs in the cytoplasm where integrase
cleaves a dinucleotide from the 3’-ends of the double-stranded viral
DNA.
The protein-DNA complex is then transported into the nucleus
where the strand transfer reaction takes place and the 3’-ends of
the viral DNA are covalently linked to the 5’-ends of the host DNA.
3’
5’
5’
5’ 3’ 5’ 3’
3’ 5’ 3’ 5’

3’-processing Strand transfer

 Integrase Inhibitors
Strand-transfer inhibitors 3’-processing inhibitors
IC50 R IC50
3P= 20.5 µM
V-104 (R=Cl)
ST= 0.059 µM
O O 3P= 0.9±0.5
O HIV-1 (IIIB) = 5.5 µM
ST= 201.3±119.7
HN NH
O-
V-165 (R=NO2)
S N O N S
O O H H 3P= 0.9±0.4
O OH ST= 16.1±0.7
Pyrano-dipyrimidines
β- Diketo acids (L-708, 906)

DNA
OH IC50
HOOC N
OH 3P= 0.4±0.5
OH
Mg2+
OH

Styrylquinolines
Mg2+

Pyrano-dipyrimidines and
Styrylquinolines inhibit 3’-processing
and display moderate antiviral activity
 Aim Of The Work
Protein engineering and
X-ray crystallography
have shown that the
mobility of the HIV-1
active site loop is
Lys 156
Loop 140-149 correlated with DNA
Lys 159
recognition.
Asn 155
Hys 67 On this basis, following
our intent to discover
integrase inhibitors
affecting the binding of
integrase with viral DNA,
we have looked for
molecules able to block
The residues involved in the interaction
with the DNA are red. The loop near
the loop in an open
the active site is represented in green. conformation.
 Conformational Analysis Of The Loop

Conformational analysis of the

loop close to the active site of
the enzyme was performed
using the software Macromodel.
50000 conformations were
generated. Among them, only
the conformations with an
energy that fell within a 10
Kcal/mol range above the
global minimum were kept.

Such conformations were submitted to cluster analysis using an

rmsd of 0.8 Å (referred to backbone atoms) and the selected ones
were used in all the following calculations.
 Pharmacophores Generation Protocol

• Analysis of the binding site by means of GRID software, taking

into account the conformations selected as previously
described.

• Identification of points of minimum in GRID molecular

interaction fields for probes C1=, C3, N1 and O.

• Design of receptor-based pharmacophoric models based on

GRID points of minimum.

• Elaboration of a virtual screening procedure based on

pharmacophoric models and docking studies.
Receptor-Based Pharmacophores
 Virtual Screening Workflow
1. Receptor
Asinex Gold Collection
based
Database – 200000 -
pharmacophoric
models
- ca. 500 -
2. Rotatable bonds 3.Evaluation
number < 10 of the
- 10 - energy of
interaction
-1- 4. Docking
studies on
5. Biological assay selected
compounds

The pharmacophoric models were in turn used to screen the

Asinex Gold Collection Database. Since Hypo1 and Hypo3 failed
to retrieve hits, several simplified 5-feature pharmacophores were
generated from the original models. About 20 000 compounds
were selected. Next, only molecules with less than 10 rotatable
bonds were kept while the molecules that poorly mapped the
pharmacophoric models (Best Fit value < 1) were removed.
 Virtual Screening Workflow
1. Receptor
Asinex Gold Collection
based
Database – 200000 -
pharmacophoric
models
- ca. 500 -
2. Rotatable bonds 3.Evaluation
number < 10 of the
- 10 - energy of
interaction
-1- 4. Docking
studies on
5. Biological assay selected
compounds

Next, the binding energy of the selected compounds was

evaluated. Each ligand identified by a particular
pharmacophore was superimposed to it and the integrase-
ligand complexes were minimized. Then, the interaction energy
between the ligands and the target was calculated through four
different scoring functions: the scoring function of Autodock 3.0
and those implemented in the XSCORE package.
 Virtual Screening Workflow
1. Receptor
Asinex Gold Collection
based
Database – 200000 -
pharmacophoric
models
- ca. 500 -
2. Rotatable bonds 3.Evaluation
number < 10 of the
- 10 - energy of
interaction
-1- 4. Docking
studies on
5. Biological assay selected
compounds

Finally, docking studies were performed on the retrieved

compounds (GOLD software). Ten compounds were chosen for
biological investigations on the basis of:
• the matching between the compound interactions and the
pharmacophoric features;
• the consensus score between pharmacophoric matching and
docking;
• the scaffolds diversity.
 Hit compound
As a results of the biological investigations, one compound (MAS
10018) was identified showing significant inhibitory potency
against IN in overall integration assay. Such compound was
also able to inhibit HIV-1 replication in cells. Further biological
studies on compound MAS 10018 have shown that it is active
both on 3’-processing and strand transfer reactions.

MAS 10018
IC50 (overall integration assay) = 9 µM
3P= 25 µM
ST= 3 µM
EC50 (IIIB)= 30 µM
EC50 (NL43wt)= 8 µM
 Binding Mode Of The Hit Compound
Loop 140-149
Green: HB acceptor
Cyan: Hydrophobic
Magenta: HB donor
Gray: Exclusion volumes
Binding Mode Of The Hit Compound
Loop 140-149

Lys 159
Val 150
Glu 152
Ile 151

Pro 142
Hys 67 Ile 141
 Conclusions

•A structure-based virtual screening protocol that taken into

account the flexibility of the loop close to the active site was
applied to discover new IN inhibitors
• As a results of the biological investigations, one compound (MAS
10018) was identified showing significant inhibitory potency
against IN in overall integration assay.
•Such compound was also able to inhibit HIV-1 replication in cells.
•Further biological studies on compound MAS 10018 have shown
that it is active both on 3’-processing and strand transfer reactions.
• Synthetic strategies are in progress to optimize the antiviral
activity of the hit compound.
 Outline

• Structure-based virtual screening for the identification

of potential inhibitors of Mycobacterium tuberculosis
thioredoxin reductase.

• Molecular modeling, synthesis and biological studies

of novel HIV-1 integrase inhibitors.

• A target for new antiviral drugs: Inhibition of the

Reverse Transcriptase Dimerization.
 Role Of Reverse Transcriptase
Reverse Transcriptase

Central role in the viral replication cycle by

generating a double-stranded DNA copy of the
single-stranded RNA genome.

Then, DNA is integrated into the host cell

genome to enable expression of viral proteins
for the production of new virions.

Reverse Transcriptase Inhibitors

Nucleoside inhibitors (NRTIs)

Non-nucleoside inhibitors (NNRTIs)

Are
Are there
there new
new possible
possible RT
RT binding
binding sites
sites as
as target
target for
for antiviral
antiviral therapy?
therapy?
 Reverse Transcriptase Structure
RT is a multifunctional heterodimer consisting of two subunits of
identical sequence named p66 p51.

p66 subunit adopts an “open” Thumb

catalytically competent p66
conformation to accomodate a RNase H
Fingers
nucleic acid template.
Palm
Thumb

Fingers

p51 subunit is closed and is

assumed to play a largely structural p51
role in the heterodimer strucuture.
 Reverse Transcriptase Structure

p66 subunit contains the catalytic site and

both polymerase and RNase H activities.

The shorter p51 subunit lacks these

functions but it is essential for loading
the p66 subunit on the template primer
(Harris et al., 1998).

Three major contact areas in the dimer interface:

Fingers subdomain of p51 Palm subdomain of p66

Thumb subdomain of p51 RNase H domain of p66

Connection subdomain of p51 Connection subdomain of p66

 RT Dimerization Process
The p66 and p51 subunits of the mature RT heterodimer are initially expressed as
part of larger polyprotein precursor (Pr160Gag-Pol).

Two models have been proposed for the formation of RT heterodimer (Sluis-Cremer
et al., 2004).

1. Concerted model Gag-Pol

In the concerted model, the two subunits

are cleaved from two separate p66 p51
precursors, followed by their association +
and assembly into the p66/p51
heterodimer.

p66/p51
 RT Dimerization Process
2. Sequential model
In the sequential model, the p66 subunit is cleaved from
Gag-Pol
separate Gag-Pol molecules, which then forms a p66/p66
homodimer intermediate, prior to formation of the p66/p51
heterodimer.
p66 p66
+ Following the formation of a p66/p66 homodimer intermediate:

p66 + p66 p66/p66 (inactive) p66/p66 (active) p66/p51

p66/p66
one of the subunits in the homodimer is then further cleaved
to form p66/p51 heterodimer RT by an HIV-1 protease. This
protease can cleave one of the two p66 subunits in the
homodimer which shows a structural asymmetry with respect
p66/p51 to RNase H domain to render this region accessible to the
protease in only one of the subunits.

Structural
Structural analysis
analysis favours
favours the
the sequential
sequential model.
model.
 RT Connection Subdomain
The first interaction between the two subunits, when the
p66 RT is forming, occurs in a TRP-rich hydrophobic cluster
Connection
located in the connection subdomain of the two subunits.

Connection subdomain: residues 312-425

Trp - - Trp Trp - - - Trp - - - Trp - - - Trp

398 401 402 406 410 414
p51

Trp402

- Mutagenesis of Trp401 and Trp414 in p66 Trp410

impairs dimerization with p51 by altering the Trp401
proper positioning of structural elements (for Asn363
example W402 and W410) in between these
residues that make contacts with p51.
Asp364
- Mutagenesis of Lys331 in p51 results in Lys331
impaired RT dimerization. Tyr405
 Aim Of The Work
The dimerization is essential for a fully functional RT and this
process offers an excellent opportunity to inhibit the enzyme by a
different mechanism by disrupting a key protein-protein
interaction.

The aim of this work was to find compounds

binding to the connection subdomain of p66
subunit and preventing the coupling between
the two subunits.

We proposed to develop structure-based pharmacophoric models

for the connection subdomain of p66 to screen a commercial
available database and applying docking studies to selected
compounds.
 p66 Pharmacophoric Models
Three different three-dimensional pharmacophoric models of the p66 connection
subdomain were generated taking into account the receptor flexibility.

1. p66 subunit was first submitted to Molecular Dynamics simulation.

p66 subunit (taken from the crystallographic structure of the

HIV-1 RT, entry 1RTH of the PDB) was first submitted to MD
calculations by means of the software package NAMD (version
2.5) with CHARMM27 force field. Hydrogen atoms were added
by means of the psfgen package.

The p66 subunit was embedded in a sphere of water molecules (60 Å radius)
applying spherical boundary conditions. The starting structure was optimized
with 1000 steps of conjugate gradient energy minimization to remove
unfavorable contacts.
 p66 Pharmacophoric Models
MD simulation was carried out at 310 K for 1 ns, collecting snapshot structures every 1
ps. The Langevin Dynamics procedure, with a dumping factor of 5 ps-1, was used to
control the temperature.
3,5

3
From MD trajectory, several K395

2,5
snapshots were chosen on the basis
2
of different conformations of relevant
RMSD

1,5

0,5
residues of the connection W410

0
0 100 200 300 400 500 600 700 800 900 1000
subdomain W402

Time (ps)
(in particular, W402 and W410).

RMS fluctuation
0,14

0,12
W402 W410
0,1

nm 0,08

0,06

0,04

0,02

0
380 385 390 395 400 405 410 415 420
Residue number

Green denotes more mobiles residues and red denotes residues

which moved less during simulation.
 p66 Pharmacophoric Models
2. For each selected snapshot, molecular interaction fields (MIFs) were computed to describe
hydrophobic interactions and hydrogen bond contacts, using three different probes (hydrophobic,
DRY; hydrogen bond donor, N1; hydrogen bond acceptor, O). The best interaction points
between the selected probes and the residues of the connection subdomain were computed.

3. The best interaction points identified by computing MIFs with DRY, N1 and O probes (left) were
converted, respectively, into hydrophobic, hydrogen bond donor and acceptor features of
pharmacophoric models. Excluded-volumes corresponding to residues W398, W402, W410 and
W414 were also added (right).
 p66 Pharmacophoric Models
4. The different structure-based pharmacophoric models, generated for the
selected MD snapshots, were merged into three pharmacophoric hypotheses
on the basis of the distance tolerance value. These hypotheses were derived
to use as a search query during the next virtual screening procedure.

Frame n. 680 Frame n. 730 Frame n. 760

Frame n. 780 Frame n. 810

Frame n. 870
 Database Virtual Screening
The ASINEX Gold Collection was screened and compounds satisfying at least one of the
pharmacophoric hypotheses and fulfilling Lipinski’s Rule of Five were retrived and docked into
the p66 connection subdomain. This approach identified ten compounds that were tested in
biological assays.

ca. 200000 cpd

Structure-based
Pharmacophoric search
ca 51000
Lipinski’s Rule of Five
filter
ca 40000
Fit value filter

110
Docking studies

10
Biological assays
 In Vitro Subunit Association Assay

The reassociation of RT subunits was initiated in the absence

or presence of MAS compounds by a 15-fold dilution into acetonitrile
Free standard buffer with a final enzyme concentration of 1.3 µM
and followed by HPLC.

Reassociation-Assay

RT Dimer RT Subunits RT Dimer

Dissociation Reassociation
+
12 % ACN
+M
AS

+
RT Subunits
 In Vitro Subunit Association Assay
Heterodimeric form of HIV-1 RT was reversibly dissociated by the addition of acetonitrile. Upon reduction of the
acetonitrile concentration to 0.8 % by a simple dilution of the samples, reassociation of RT was initiated and
followed by size-exclusion chromatography or increase of the enzymatic activities.

O
H 3C OH
N N N
Et
N
NH2 Applying this procedure, MAS0 and MAS1
O N N N Et S
CH3 O
showed a dose-dependent inhibition of the
dimerization process. MAS0 proved to be about
MAS0 OH
MAS1 5 times more active than MAS1 in this assay.

b) Dose-dependent inhibition of RT
reassociation by MAS compounds. Data
were fitted to a hyperbolic equation. 50%
inhibition was observed at ca. 150 µM for
MAS0 (○) and ca. 750 µM for MAS1 ( ).
Prior to HPLC size-exclusion
chromatography samples were incubated
for 16 h at 20ºC.

a) Reassociation reaction of acetonitrile

treated HIV-1 RT heterodimers in the
absence (□) or presence of MAS0 (○)
and MAS1 ( ) compounds (1 mM each).
Inhibition of RT Activities by MAS0

A) Dose-dependent, simultaneous inhibition of both

the polymerase (○) and the RNase H activity ( ) by
MAS0, yielding IC50 values of 155 and 111 µM,
respectively.

B) Effect of RT/MAS0-preincubation time on

polymerase (○) and the RNase H activity ( ).
Inhibition of RT could only be observed following a
preincubation of enzyme and MAS0 with t1/2 of about
2 h. During preincubation no significant reduction of
enzymatic activities was observed in the absence of
the compound (data not shown).
 Specificity of inhibition by MAS0
To analyze the specificity of MAS for HIV-1 enzyme, polymerase assays
were performed with HIV-2 RT, Avian Myeloblastosis Virus (AMV) RT, and
E. coli DNA Pol I (Klenow fragment, KF).

MAS0 decreased the polymerase activity

of HIV-2 by about 60%, whereas the
activity of the AMV RT and KF remained
unaffected.

The different levels of inhibition could be explained by some minor sequence variation
in this region. The likewise heterodimeric AMV RT, on the other hand, is not affected
by MAS0.
Even though all three RTs (HIV-1, HIV-2 and AMV) do presumably share a similar
overall folding (the X-ray structure of AMV RT is not known), only enzymes
containing a tryptophan cluster (AMV RT has no such motif) are affected.
 Conclusion

Taking into account biological assays

results, MAS0 is supposed to interfere
with the dimer equilibrium by shifting the
equilibrium from an active to an inactive
dimer by trapping the enzyme in the
inactive conformation.

As the structures of the intermediate inactive dimer and the

monomeric species are not known, the structures shown here for
illustration purposes are all derived from the structure of an active
heterodimer.

This study represents the first successful rational screen for a small
molecule HIV RT dimerization inhibitor, which may serve as an attractive hit
compound for the development of novel therapeutic agents.
 Acknowledgments

• University of Leuven, Belgium: Myriam Witvrouw

Zeger Debyser

• Universität Lubeck, Germany : Tobias Restle

Dina Grohmann

• MOLISA GmbH, Magdeburg: Leopold Flohé

Timo Jaeger

We thank Gabriele Cruciani for the access to the GRID code

 at t en ti on ! !!
r y o u r ki n d
Th a n k s f o
 From Eastern Roman Empire…

…to the Western one

 Arrivederci in Siena:

VII EWDD
Seventh European Workshop
in Drug Design
May 24 - 30, 2009 – University of Siena (Italy)

www.unisi.it/EWDD