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Doctoral Thesis
Novel ligands for the neuronal nAChRSynthesis, in vitro characterizations and binding models
Author(s): Bisson, William Henry
Publication Date: 2003
Permanent Link: https://doi.org/10.3929/ethz-a-004624980
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ETH Library
Diss. ETH Nr. 15220
Novel ligands for the Neuronal nAChR:
Synthesis, in vitro Characterizations and
Binding Models
A dissertation submitted to the
Swiss Federal Institute of Technology Zurich
for the degree of
Doctor of Natural Sciences
presented by
William H. Bisson
Laurea in Chimica
University of Padua, Italy
born April 28th, 1974
citizen of Treviso, Italy
accepted on the recommendation of
Prof. Dr. P.A. Schubiger, examiner
Prof. Dr. G. Folkers, co-examiner
Dr. G. Western, co-examiner
2003
I
TABLE OF CONTENTS
Table of contents
List of Abbreviations
Summary
Zusammenfassung
CHAPTER T_^ ^_^
Introduction
The Nicotinic Acetylcholine Receptor
The non opioid alkaloid Epibatidine
Pharmacophores
Binding Studies
Electrophysiology Studies
Molecular Modeling
Molecular Docking
Aim ofthe Project studied in this Thesis
CHAPTER II
Synthesis and binding studies evaluation of epibatidine analogues as novel
ligands for the nicotinic acetylcholine receptors
Abstract
Introduction
Result and Discussion
Experimental
Conclusions
References
CHAPTER HI
Affinity and electrophysiological evaluation of epibatidine and cytisine analogues
as novel ligands for the nicotinic acetylcholine receptors
Summary
Introduction
Result and Discussion
Experimental
Significance
Acknowledgments
References
CHAPTER IV^
Homology Modeling, Dynamics of Rat and Human Extracellular Domain of a4ß2and a7 Neuronal Nicotinic Acetylcholine Receptor Subtypes
Abstract
Introduction
Methods
Results and Discussion
Conclusions
References
CHAPTER V
Molecular docking to investigate ligand selectivity for the acetylcholine bindingsite of the rat a4ß2 and a3ß4 nicotinic subtypes
Abstract
Introduction
Ill
Material and Methods
Results and Discussion
Conclusions
References
CHAPTER VI
Outlook
Publications and Presentations
Curriculum Vitae
Acknowledgments
rv
LIST OF ABBREVIATIONS
ACh Acetylcholine
AChBP Acetylcholine binding protein
AD Alzheimer's disease
AMBER Assisted model building energy refinement
Boc Butyloxy-carbonyl
Cdna Cyclic DNA
CNS Central nervous system
2-D Bidimensional
3-D Tridimensional
DAST Diethylamino sulfurtrifluride
DclEpi Dechloroepibatidine
DCM Dechloromethane
DMF Dimethylformamide
Epi Epibatidine
EPB Epibatidine
ESI Electron spray ionization
EtOAc Ethylacetate
Et3N Triethylamine
GABA Gamma-amino-butyrric-acid
HEPES N-2-hydroxyethylpiperazine-N'-2-ethane-sulphonate
HSV1 TK Herpex simplex virus 1 tymidin kinase
5-HT Serotonin
IC50 Ligand concentration required to displace 50% ofradioligand
binding
Kd Dissociation constant
Ki Inhibition constant
LD50 Median lethal dose
LGIC Ligand-gated ion channels
MD Molecular dynamics
MeOH Methanol
Met Methyl
V
MS Mass spectrometry
MW Molecular weight
NAChRs Nicotinic acetylcholine receptors
NMR Nuclear magnetic resonance
PDB Protein data bank
PET Positron emission tomography
Ps Picosecond
QSAR Quantitative structure activity relationship
RMSD Root mean square deviation
SE Standard error
tl/2 Half time
TFA Trifluoroacetic acid
THF Tetrahyrofuran
TLC Thin layer chromatography
UV Ultraviolet
VI
SUMMARY
The present project is aimed at the design and development of non-toxic ligands with
specificity and subtype selectivity towards the neuronal nAChRs. The potential benefit
of nicotinic ligands for a variety of neurodegenerative diseases (like AD), due to the
involvement of nAChRs in cognitive processes, has energised research efforts in this
direction.
The natural ligands (-)-epibatidine and (-)-cytisine proved to exhibit high affinity and
agonism activity towards neuronal nAChRs but their clinical use, cause of their
toxicity, is precluded.
O
(-)-epibatidine (-)-cytisine
A series of epibatidine and cytisine derivatives were synthesised and investigated by in
vitro binding and electrophysiology studies. This permitted to put into correlation the
ligand chemical structure and the affinity and biological response towards different
nAChR subtypes.
Binding-experiments on the two most present subtypes in the brain, were performed
using the competition assay with [3H]-cytisine (oi4p2 nAChR) and [ l]-alpha-
bungarotoxin (0,7 nAChR) as radioligands.
Voltage clamp electrophysiology was performed on neuronal (0^2 and 017) and
ganglionic (a3ß4) nAChRs reconstituted in Xenopus oocytes.
All compounds showed, by binding studies, low affinity towards the oc7 neuronal
nicotinic receptor and much higher affinity for the a4ß2 subtype, which indicates high
selectivity towards the 0^2 neuronal subtype.
VII
The affinity for oc4p2 neuronal nicotinic receptor is related to the chemical structure of
the ligand. All compounds synthesised possess the three geometrically independent
pharmacophoric structural features requested for binding recently mentioned by
Nicolotti et al. Nevertheless the affinity obtained varied a lot among the compounds
studied. Bromocytisine showed the highest affinity (K; = 0.153 nM) towards the a4p2
subtype but low subtype selectivity due to its full agonism towards the ganglionic 0^4
subtype receptor. Compound (±)-33bMet, shown below, exhibited high affinity (Kj = 2
nM) and partial agonism for the a4ß2 subtype but significant subtype selectivity too.
Compound (±)-33bMet was then injected intravenously into mice and was proved to
be relatively non-toxic with a median lethal dose of> 0.5 mg/kg mouse body weight.
All the binding, electrophysiology and toxicology data found make compound (±)-
33bMet to be a potential radiotracer for in vivo brain PET investigations.
Recently the crystalline structure of the acetylcholine-binding protein (AChBP),
isolated from snails, has been resolved. It is a soluble homopentamer protein revealing
in the binding site region high homology with the extracellular N-terminal domain of
different species nAChR subunits. This would give then the possibility to model some
ofthe existing nAChR subtypes.
In the present work the homology-models of the extracellular domain of the rat and
human neuronal (o4p2) and (017) and of the rat ganglionic (a3ß4) nAChR subtypes were
built, energetically minimized and their stability was assessed using molecular
dynamics.
A series of nicotinic ligands were docked into the binding cavity of the non-
dynamicised rat 0^2 and 0^4 subtypes to investigate the relationship between
structure and affinity and between structure and subtype selectivity, which is probably
connected to toxic side effects.
vm
The docking in both modeled subtypes confirmed, for the protein-ligand complex, the
importance of cation-7i interactions between the quaternary positive nitrogen of the
ligand and the aromatic pocket in the binding site region and the hydrogen bond
between a hydrogen acceptor in the ligand and a hydrogen donor of a residue present
in the receptor binding site.
In the rat 01^2 subtype two docking orientations (clusters 1 shown in the Figure below
and 2), with the highest consensus score, were identified involving high affinity
ligands like epibatidine, dechloroepibatidine and (S)-A-85380. The docking
orientations clusters 1 and 2 are preferred respectively by clinically toxic and non-toxic
ligands, which turned to be an interesting result.
All ligands studied didn't dock well into the a3p4 model with the exception of
epibatidine, dechloroepibatidine, (±)-33bMet and the Abbott derivative (S)-A-98795.
The docking data obtained reveal that the presence of the oxygen atom between the
pyridine ring and the aliphatic moiety plays an important role in the 0^2 subtype
selectivity and that the flexibility constriction around the ligand C-O-C and C-C-C
angles increases affinity towards the a3p4 cavity.
These docking investigations showed that the studied nicotinic models are useful for
structure-based design of specific and selective 04p2 nAChR subtype ligands for
therapeutic and diagnostic applications in neurological diseases.
IX
ZUSAMMENFASSUNG
Das vorliegende Projekt hat zum Ziel, nicht-toxische Liganden zu entwerfen und zu
entwickeln, die Spezifität und Subtyp-Selektivität gegenüber neuronalen nAChRs
aufweisen. Der potentielle Nutzen von nicotinischen Liganden für eine Vielfalt von
neurodegenerativen Erkrankungen (wie beispielsweise der Alzheimer-Krankheit),
herrührend durch die Beteiligung von nAChRs in kognitiven Prozessen, hat die
Forschung auf diesem Gebiet angetrieben.
Die natürlichen Liganden (-)-Epibatidin und (-)-Cytisin wiesen eine hohe Affinität
sowie agonistische Aktivität gegenüber neuronalen nAChRs auf, deren klinischer
Gebrauch ist jedoch wegen deren Toxizität ausgeschlossen.
H2N+
(-)-epibatidine (-)-cytisine
Darauffolgend wurde eine Reihe von Epibatidin- und Cytisinderivaten synthetisiert
und in vitro mit Hilfe von Bindungs- und elektrophysiologischen Studien untersucht.
Dies erlaubte, die chemische Ligandstruktur, die Affinität und die biologische Antwort
auf verschiedene nAChR-Subtypen in Zusammenhang zu bringen. Bindungsstudien
der beiden am häufigsten vorkommenden Subtypen im Gehirn wurden unter
Anwendung des Competition Assays mit [3H]-Cytisin (a4ß2 nAChR) und [125I]-alpha-
bungarotoxin (a7 nAChR) als Radoliganden durchgeführt. Des weiteren wurden
Voltage-clamp-Elektrophysiologiestudien an neuronalen (cl$2 und (X7) und
ganglionären (a3ß4) nAChR von Xenopus Oocyten vorgenommen.
Alle Verbindungen zeigten in den Bindungsstudien niedrige Affinität gegenüber des
neuronalen a7-nikotinischen Rezeptors, aber weit höhere Affinität gegenüber dem
X
neuronalen a3ß4-Subtyp, was für eine hohe Selektivität des neuronalen a4ß2-Subtyps
spricht.
Die Affinität gegenüber dem neuronalen a4ß2-nikotinischen Rezeptor steht in
Verbindung mit der chemischen Struktur des Liganden. Alle synthetisierten
Verbindungen besitzen die drei geometrischen, voneinander unabhängigen
pharmakophoren Strukturmerkmale, welche für die Bindung notwendig sind und die
kürzlich von Nicolotti et al erwähnt wurden. Allerdings variierten die erhaltenen
Affinitäten stark bei den betrachteten Verbindungen.
Bromocytisin zeigte die höchste Affinität (K = 0.153 nM) gegenüber dem cl$2-
Subtyp, jedoch geringe Subtyp-Selektivität wegen seines Agonismus gegenüber dem
ganglionären a3ß4-Subtyp-Rezeptor. Verbindung (±)-33bMet, wie unten gezeigt, wies
gegenüber dem a3ß4-Subtyp hohe Affinität (K; = 2 nM) und partiellen Agonismus
sowie signifikante Subtyp-Selektivität auf.
Verbindung (±)-33bMet wurde im Anschluss daran intravenös in Mäuse injiziert und
erwies sich als relativ untoxisch mit einer mittleren letalen Dosis von >0.5 mg/kg
Maus-KÖrpergewicht.
Alle gefundenen Bindungs-, Elektrophysiologie- und Toxiziätsdaten belegen, dass
Verbindung (±)-33bMet ein potentieller Radiotracer für in vivo PET-Untersuchungen
am Gehirn ist.
Kürzlich wurde die Kristallstruktur des Acetylcholin-Bindeproteins (AChBP), isoliert
aus Schnecken, entschlüsselt. Es handelt sich um ein lösliches, homopentameres
Protein, welches in seiner Bindungsseite eine hohe Homologie mit der extrazellulären,
N-terminalen Domäne der nAChR-Untereinheiten verschiedener Spezies aufweist.
Dies würde gegebenenfalls ermöglichen, einige der im Körper existierenden nAChR-
Subtypen zu modellieren.
XI
In der vorliegenden Arbeit wurden demzufolge Homologie-Modelle der
extrazellulären Domäne der ganglionären (a3ß4)-nAChR-Subtypen in der Ratte und der
neuronalen (a4ß2)- und (ci7)-nAChR-Subtypen der Ratte und des Menschen erstellt,
energetisch minimiert und ihre Stabilität mit Hilfe von molekularer Dynamik geprüft.
Eine Reihe nikotinischer Liganden wurden in die Bindungstasche der nicht-
dynamisierten a$2- und a3ß4-Subtypen der Ratte eingepasst, um die Beziehung
zwischen Struktur und Affinität sowie zwischen Struktur und Subtyp-Spezifität zu
studieren, welche möglicherweise mit toxischen Nebeneffekten in Verbindung steht.
Das Docking in beiden modellierten Subtypen bekräftigte für den Protein-Ligand-
Komplex die Wichtigkeit der Kationen-71-Interaktionen zwischen dem quaternären
positiven Stickstoffatom des Liganden und der aromatischen Tasche in der
Bindungsregion und der Wasserstoffbrücke zwischen einem Wasserstoff-Akzeptor im
Liganden und einem Wasserstoff-Donor einer Amionsäure, die in der
Rezeptorbindungsstelle vorhanden ist. Im 04p2-Subtyp der Ratte wurden zwei
Docking-Orientierungen (Cluster 1 und 2) mit dem höchsten Consensus Score
identifiziert, die hochaffine Liganden wie Epibatidin, Dechloroepibatidin und (S)-A-
85380 miteinbeziehen. Die Docking-Orientierung Cluster 1 wird von klinisch
toxischen und die Docking-Orientierung Cluster 2 von nicht-toxischen Liganden
bevorzugt, was sich als interessantes Resultat herausstellte.
Alle der studierten Liganden dockten nicht gut ans a3ß4-Modell mit Ausnahme von
Epibatidin, Dechloroepibatidin, (±)-33bMet und vom Abbott Derivat (S)-A-98795. Die
erhaltenen Docking-Daten zeigen, dass die Anwesenheit vom Sauerstoffatom zwischen
dem Pyridin-Ring und dem aliphatischen Teil eine wichtige Rolle in der <x^2-Subtyp
Selektivität spielt und dass die Flexibiltätseinschränkung um die C-O-C- und C-C-C-
Winkel des Liganden die Affinität gegenüber der a3ß4-Tasche erhöht.
Diese Docking-Untersuchungen zeigten, dass die untersuchten nicotinischen Modelle
hilfreich für Struktur-basiertes Design von spezifischen und selektiven a4ß2-nAChR-
Subtyp Liganden sind, die für therapeutische und diagnostische Zwecke bei
neurologischen Erkrankungen Anwendung finden.
1
CHAPTER 1
Introduction
THE NICOTINIC ACETYLCHOLINE RECEPTOR
Nicotinic acetylcholine receptors (nAChRs) are trans-membrane oligomeric proteins,
part of the family of the ligand gated ionic channels which comprises also GABAA,c,
glycine and 5-HT3 receptors (1,2). They are widely distributed in the mammalian
organism, located at the neuromuscular junction, autonomic ganglia and at the central
nervous system. Recently they have been also localized in human bronchial epithelial
and endothelial cells (3).They mediate acetylcholine neurotransmission (4), and after
binding, they have excitatory effects, by the influx of cations through the open
channel, which causes dcpolarisation of the postsynaptic membrane, propagating the
electrical activity from neuron to neuron or from neuron to effector cell (5). The first
nAChRs characterised were the ones expressed by vertebrate neuromuscular junction
and Torpedo Marmorata (picture).
The receptor is an assembly of five subunits forming a trans-membrane pentamer with
the stoechiometry a2,ß,y,o (6). This composition would correspond with the vertebrate
embryonic neuromuscular junction receptor which replaces the y subunit by the c
subunit in the adult form. According to these studies the nAChRs were also localised
in the central nervous system (7).
Additional subdivision between subunits was made according to specific residues
found in the ACh-binding site region in the N-terminal domain. For example the
presence of two adjacent cysteines classified muscular and neuronal a subunits.
At the present time nine a (a2- alO) and three ß (ß2- ß4) neuronal nAChR subunits
have been cloned (8,9). Neuronal nAChRs are mostly composed by a4 and ß2 subunits
2
and for a minor part of a7 subunits. Site-directed mutagenesis and single-channel
studies have demonstrated that the a4ß2 nAChRs are composed of two a4 and three ß2
subunits forming heteromeric receptors while 017 nAChRs arc homomeric receptors
made up of 5 07 subunits.
ïi t-t-
1I1/*]
-—- A
'
# i- -—-v
"S
\-~~_J----fü Ai i O
</
^'* ^_^j; ;
(
'"l i. M*xt: }~ 1 m
Oil,
vwVI»' '"'l'a.
•lui.,
^ «
1' 3 } î| 's' Tï
n „( </41
s
Figure 1. Neuromuscular nAChR subtype. Subunit compositionof the embryonic neuromuscolar nAChR subtype.
The pentamer assembly forms the pore which is permeable to cations like Na,K and
Ca2+ in the way shown in Figure 1. Different subunit compositions characterise then
the difference existing nAChRs subtypes present in the mammalian organism.
Neuromuscolar and neuronal a4ß2 heteromeric nAChRs have two ACh binding sites
while neuronal a7 homomeric nAChRs have five ACh binding sites ( 10).
High-resolution electron microscopy techniques revealed the location of the ACh
binding site approximately 30 A° above the membrane surface in the extracellular N-
terminal domain of the transmembrane protein (11). But only recently with the
resolution of the crystal structure of the acetylcholine binding protein (AChBP)
isolated from snails confirmed the pentameric organization of the N-terminal domain
and the localization of the ACh binding site at the interface between an a subunit and
an adjacent subunit as in Figure 2(12).
3
heteromeric receptor
IVincipalV
component \\
Loop A
I oop H
r
"—— VIS *-*'*!
a
IaW}> C \ ,
/ tompk'menlaiy
/ component
"El72
ß
Figure 2. The a4p2 heteromeric neuronal nAChR subtype binding site. Subunit
composition and localization of the binding sites (liier ct al. 2001).
Ligand-gated channels, like nAChRs, behave as allosteric proteins (with a cooperative
way between the different binding sites on the same receptor) with multiple
conformations and the receptor can change state in the absence of the ligand. The
"multiple allosteric states" describes four B, A, I and D states (see Figure 3). In the
absence of an agonist the receptor is in the resting B (closed) state although a fraction
^A
Figure 3. The multiple allosteric states system. Acilive state (A),
Basal state (B), Intermediate state (1) and desensitised state (D).
of the receptors can be in the desensitised D (closed) state according to the equilibrium
constants. When the agonist binds, the active A (open) state is stabilised first, then the
4
intermediate I (closed) state and finally with a slower time constant, the desensitised D
(closed) state (13).
The identification of central nervous system nAChRs opened a way to study
neurotransmission but their anatomic location in the brain regions (neocortex,
hippocampus) involving cognitive effects, like memory and learning (4,14), raised the
interest of neuroscientists to investigate the correlation between neurological diseases
like Alzheimer's Disease (AD) and nAChRs (15,16). The positive cognitive and
neuroprotective effects of nicotine as therapeutic agent (17,18) and the potential of A-
85380 (19,20) as a diagnostic Positron Emission Tomography (PET) agent enhanced
the efforts, with in vitro and in vivo characterizations, to find high affinity, subtype
selective and non toxic ligands for the neuronal nAChRs.
THE NON OPIOID ALKALOID EPIBATIDINE
History
In 1974 John Daly and co-workers of the National Institute of Health isolated from
skin extract, of the poison frog Epipedohates tricolor (picture) living in the Pacific
Ecuadorian highlands, a compound which was called alkaloid 208/210 (its MW from
mass spectrometry) (21). Daly demonstrated that this new alkaloid, when injected
subcutaneously into mice, was a potent analgesic measured in the Straub-tail response
(22).
One milligram of extract were obtained from the skins of 750 frogs! This complicated
then the structure elucidation of the substance due to the limited techniques present in
that time for such a small amount. But in 1991 in Daly lab using more sensitive and
sophisticated nuclear magnetic resonance the structure of the substance was
determined and named epibatidine, (1R, 2R, 4S exo-2-(6-chloro-3-pyridyl)-7-
azabicyclo[2.2.1]heptane) (23) as shown in Figure 4.
4
Figure 4. Epibatidine. Chemical structure of epibatidine.
Pharmacological properties
Epibatidine was shown to be a potent analgesic (about 200 times more potent than
morphine), but what was found interesting is its non-opioid mechanism of action.
Characteristic opioid effects were observed after mice injection but those effects were
not attenuated by pre-treatment with the opioid receptor antagonist naloxone (23). So
the question became if epibatidine analgesic doesn't involve opioid receptors, how
does it produce then the pain relief? Daly and other research groups answered this
question by examining the interaction of epibatidine with nicotinic acetylcholine
receptors (nAChRs). Epibatidine was found to bind and activate central nervous
system nAChRs very well (K; = 0.043-0.055 nM) and the analgesic effects of
epibatidine were blocked by mecamylamine (a nicotinic receptor antagonist).
Compared to S-(-)-nicotine (Ki =1-2 nM), the naturally occurring stereoisomer, which
has about 20-fold higher affinity than its enantiomer, both enantiomers of epibatidine
((-)-enantiomer is the natural epibatidine) showed to have high affinity for central
nervous system nAChRs. Both enantiomers of epibatidine bind well to the ganglionic
peripheral nervous system nAChRs too (24,25). This was found to be the reason of the
side effects as marked hypertension observed with the in vivo use of epibatidine as an
analgesic (26). Unfortunately the therapeutic and PET diagnostic use of epibatidine in
clinical medicine is precluded by its high toxicity but the high affinity and agonism of
this compound toward the central nervous system nAChRs (25) opened a new way to
study these ligand gated ionic channels and the related neurological diseases.
6
PHARMACOPHORES
The potential benefit in a variety of neurodegenerative diseases involving the CNS of
neuronal nicotinic acetylcholine receptor (nAChR) subtype selective ligands (27)
increased the scientific effort to investigate the nicotinic pharmacophores (10). Since
forty years several investigations have been made in this direction to define the
possible pharmacophoric groups for nicotinic ligands. In 1970 Beers and Reich (28) in
their "Pharmacophore Model" postulated that nicotinic agents are characterised by an
onium moiety and a hydrogen bond acceptor atom and the distance between them
should be around 5.9 Â. Then after 16 years in the 1986 Sheridan and co-workers,
studying nicotinic agonists like (S)-nicotine and (-)-cytisine, modified Beers and
Reich feature's postulation by introducing the "three-point Pharmacophore Model"
(Figure 5) using the Distance Geometry Approach (29). The three points are referring
to a basic nitrogen atom (A), a hydrogen bond acceptor atom like the nicotine pyridine
nitrogen or the cytisine carbonyl oxygen (B) and a dummy atom representing for
example the center ofthe nicotine pyridine ring or the cytisine carbon carbonyl.
+
C4.0Ä
AN+
Figure 5. Three-point Pharmacophore Model. Scheme of the
Three-point Pharmacophore Model.
Barlow and Johnson in 1989 challenged the concept of the Sheridan pharmacophore
model based on X-ray crystallographic data of (S)-nicotine and (-)-cytisine (30). They
suggested that agonist activity requires a charged nitrogen (i.e. an onium site) and a
planar area on the receptor able to recognize an aromatic ring or a double bond through
Ti-electron or hydrophobic interactions. This "point plus flat area" concept seemed to
account better than the "three-point Pharmacophore Model" for a wider range of
nicotinic agonists.
7
Other relevant contributions have been made in the last years. Manallack et al. in 1996
(31 ) analysing semirigid agonists proposed as pharmacophoric features a cation (I), a
center of a pyridine ring or the carbon atom of a carbonyl group (II) and a dummy
atom representing a functional group, localised in the receptor region, able to establish
hydrogen bonds with a pyridine nitrogen or a carbonyl oxygen (HI).
Recently Tonder et al. (32) reported possible pharmacophoric characteristics for an
agonist toward the a4ß2 subtype receptor which should sum up all that was published
during the last 40 years. Three site points: i) protonated nitrogen atom, ii)
electronegative atom capable of forming hydrogen bonds and iii) the centre of a
heteraromatic ring or of a C=0 bond. These three points must be within a certain range
of inter-atomic distances and angles.
Unfortunately despite the efforts done, all mentioned concepts suffered from some
limitations such as the low number of nicotinic ligands investigated (33). Nicolotti et
al. proposed then three geometrically independent key structural features: i) a
positively charged nitrogen atom for ionic or hydrogen bond interactions, (ii) a lone
pair of the pyridine nitrogen or a specific lone pair of carbonyl oxygen, as a hydrogen
bond acceptor, and (iii) a centre of a hydrophobic area generally occupied by aliphatic
cycles.
These features were then analysed and confirmed by using predictive 2-D and 3-D
quantitative structure activity relationship (QSAR) investigating 270 nicotinic agonists
of various chemical classes (34).
This seems to represent a valuable computational tool in the design of new nAChRs
agonists with therapeutic and diagnostic potential but the recent resolution of the
crystal structure of the acetylcholine binding protein (AChBP) (12) (Figure 6) which
can be used as a template for the extracellular N-terminal domain of nAChRs (35)
opened new possibilities with modern technologies of Molecular Modeling and
Docking to investigate and well define the pharmacophores ofthe nicotinic ligands.
8
Figure 6. AChBP monomer binding site. Plus side on the left and
minus side on the right (Brejc et al 2001).
BINDING STUDIES
During the past 25 years, the radioligand-binding technique has become an important
tool to study the affinity of compounds toward brain receptors. The membrane receptor
assay is the most frequently used assay based on this technique. There are different
basic experiments that are performed using this membrane assay system. The
"inhibition experiment" was the one used in the present project. Here the receptor
concentration, the radioligand concentration, and time are all constant, while the
concentration of the unlabeled or inhibiting drug is variable (36). In the case of
neuronal nAChRs the radioligands used were [3H]-cytisine (ti/2 = 12 years) for the a4ß2
subtype (37) and [125I]-a-bungarotoxin (ti/2 = 60 days) for the a7 subtype (38). A
typical inhibition curve is shown in Figure 7.
The concentration required to displace 50% of ligand binding at the radioligand
concentration used (IC50) and inhibition constant (Ki) values of many unlabeled drug
can be obtained from these experiments. During the years different synthesised
compounds were tested using the inhibition experiment with [ H]-cytisine and [ I]-
alphabungarotoxin to find out respectively the affinity toward a4ß2 and a7 nAChRs, the
two most present subtype in the brain (39-40). Other radioligands like [3H]-epibatidine
(ti/2 = 12 years) toward a4ß2 and [125I]-MLA (ti/2~ 60 days) toward <x7 nAChRs have
been also used (41).
9
Figure 7. Inhibition curve. On the y-axis the percentage of radioligandbound to the membrane; on the x-axis the log value of the concentration
of the unlabeled drug.
The inhibition binding assay is definitely an useful method to find compounds affinity
toward specific brain receptor subtype due to the possibility of exploiting specific
subtype radioligands.
ELECTROPHYSIOLOGY STUDIES
A major goal of electrophysiology is to understand how an ion channel functions at a
molecular level. The recent years advances in molecular biology (protein expression,
cloning) and electrophysiology (patch clamp) produced a powerful tool to study ion
channel receptors.
In 1983 Gundersen et al.(42) and Miledi et al.(43) were the first to demonstrate that a
variety of receptors and channels from the central nervous system could be expressed
in Xenopus frog oocytes (picture).
10
At the present time nine a (a2- a 10) and three ß (ß2- ß4) neuronal nAChR subunits
have been cloned (8,9). Combined with reconstitution techniques either in Xenopus
oocytes or in stable transfected cell lines this offers the possibility to express in host
systems different nAChR subtypes to be to studied by electrophysiology in detail with
high specificity (44,45).
In the present project the used method is the following:
(a) (b) (c)
(a) isolation of the Xenopus oocyte;
(b) intranuclear injection of oocyte with cDNA expressing the wanted nicotinic
subtype;
(c) two-electrode voltage clamp recording the ion current signal due to the channel
opening by the ligand application.
The application of an agonist records an inward ion current due to the opening of the
channel and the depolarization of the oocyte cell membrane expressing the nAChR
subtype giving a typical current signal (Figure 8).
A,n bet - U"J
mM 1*1 (.V m > , ["!»|
1
i
1 f
t L i RH^
•
A *•
<% à t
rnM 'ITOliM.
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9
Figure 8. Typical current signal. Current evoked by a saturating ACh test pulse at
the a4ß2 nAChR are compared to (+)-DClEpi and (-)-DClEpi (Spang et al. 2000).
11
The investigation of receptor response of neuronal nAChRs and their sensitivity to
agonists and antagonists is an important application of voltage clamp
electrophysiology (46,47) and in combination with affinity measurements it is still a
powerful tool in the development of ligands toward specific brain nAChR subtypes.
MOLECULAR MODELING
Molecular modeling is a powerful methodology for analyzing the three dimensional
structure of biological macromolecules (48). Many times molecular modeling
integrates with NMR spectroscopy and X-ray crystallography structure
characterizations. NMR and X-ray methods generate co-ordinate data that are used to
be deposited in the Protein Data Bank (PDB) (49, http://www.rcsb.org/pdb/). The
number of macromolecules in the database is rapidly growing (from 1000 in 1990 to
14000 in 2000) and the fact that the database structures are accessible to everybody,
will certainly improve scientific effort to have more knowledge about protein folding
and function. An example of getting a macromolecular structure from the PDB system
is shown in Figure 9 which shows the 2.70 Â resolution X-Ray structure of the
homopentameric Acetylcholine Binding Protein (AChBP) from Brejc et al. (12).
Figure 9. AChBP. X-Ray Structure of the Acetylcholine
Binding Protein.
12
In recent years sequence homology alignment and model building techniques are
improving faster than NMR or X-Ray crystallography protein structure determinations.
This would be of great benefit for the many existing important proteins with amino
acid sequence available but without known three-dimensional structure.
The Swiss-Model Program (50,51 http://www.expasy.ch/swissmod/SWISS-
MODEL.html), for example, provides different approaches in this direction but of
course expertise is required to judge the results obtained. An example of triple-
sequence homology alignment is illustrated in Figure 10.
r~ n <~' -f'"
ir '"' |! 1»"~" i l( ''it j ~^r' n
t i jtu- ml mu M i |- w jn- jii , ji' "L.fu ^ <„ -L*. "' '- _J"L-. -t_. uJ ,. _i« J" L iJi i__ „—J >U
'L'< <-^
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i- LJiuJfP_J> ".lU'iU ii L_-„if _«iiiri I—__._ rr iLJ
'I-
FigurelO. Homology alignment. An example of triple-sequence
homology alignment.
For the model construction, special programs like SYBYL (Tripos Inc., St. Louis,
USA, http://www.tnpos.com) and Insightll (MSI, San Diego, USA,
http://www.accelrys.com) are worldwide used and they also provide the possibility of
model geometrical corrections caused for example by cis-configuration peptides or
amino acid D-chiralities.
The protein model water boxing, charge neutralization, energy minimization,
Molecular Dynamics (MD) and RMSD (root mean square deviation) graphics versus
time and versus residue number are performed using the software package AMBER
(Assisted Model Building with Energy Refinement) provided by University of
California (http://www.amber.ucsf.edu/).
Figure 11 represents an example of a protein model water boxed and neutralized by
counter ions.
13
To simply check the geometry of the model protein bonds, bond angles, torsion and
residue chirality etc. the program PROCHECK (52) is often used giving you the
possibility to follow the protein model geometry situation during every energy
minimization and dynamic step.
The recent resolution of the crystal structure of the acetylcholine binding protein
(AChBP) (12) shown in Figure 9 opened the possibility with Molecular Modeling to
build homology
Figure 11. Molecular modeling. Molecular modeling of a protein-
protein complex and counter ions (space filling representation) alongwith water molecules (red and white line drawing representation)
ready to undergo Molecular Dynamics (MD) (Forster et al. 2000).
models of the extracellular N-terminal domain of different nAChRs subtypes (35,53)
which will be useful to study this receptor of which, unlike of some other ion-channel
receptors, tridimensional structure is not yet known.
Molecular Modeling with the future growth of protein structure databases will
certainly become a valuable tool for a better understanding of structural biology.
MOLECULAR DOCKING
The growth of protein structure databases and the recent advances in combinatorial
chemistry, which enables chemists to synthesize a very large number of compounds,
14
made Molecular Modeling and Molecular Docking to a wide range of proteins
possible.
An important and useful area of application of Molecular Modeling is the investigation
of the interactions of the complex protein-ligand (48,54). A fast development of
pharmaceutical drugs in clinical studies and the high costs of organic synthesis require
valuable screening methodologies for drug design and Molecular Docking may be a
solution to this problem. The study of modeled protein-ligand complexes (Figure 12) is
able to identify the site of ligand-binding and the geometry of the complex and to
investigate the free energy of binding. The selection of favourable ligands builds up
libraries of compounds against the modeled protein and this is and will be an important
approach to drug design and discovery (48,55).
Figure 12. Molecular docking. Docking cluster of aciclovir (sticks)
and aciclovir crytstal structure binding mode (balls and stcks) into
the HSV1 TK binding site (Pospisil et al. 2002).
Virtual screening methods by computational docking have two important issues:
docking and scoring. Different database docking scoring programs used in
combination with different scoring functions are being used (48,56). In the present
project the SYBYL docking program FlexX (Tripos Inc., St. Louis, USA,
http://www.tripos.com) in combination with the consensus scoring function Cscore
(http://www.tripos.com/software/cscore_ print.html) was used (57). FlexX rapidly
15
docks a flexible ligand into a rigid binding site (~ 3 min per compound) as shown in
Figure 13.
CScore creates columns in a Molecular Spreadsheet that report raw scores for each
scoring function. CScore also creates a consensus column, containing integers that
range from 0 to the total number of scoring functions. Each complex whose score
exceeds the threshold for a particular function adds one to the value of the consensus;
configurations below the threshold contribute a zero. CScore offers multiple
approaches for the evaluation of ligand-receptor interactions. The strengths of
individual scoring functions combine to produce a consensus that is» both more robust
and more accurate than any single approach currently in use.
The application of computational docking requires the tridimensional structures of the
protein binding site but in the recent years some programs have been used to dock
compounds into protein without prior knowledge of the binding site location and
conformation (59).
With the time going on the role of computer-aided prediction and design of active
compounds through the investigation of protein-ligand interactions with Molecular
Docking will increase even more its importance in the field of pharmaceutical
research.
Figure 13. AChBP binding site region. Docking ofacetylcholinein the AChBP binding site region by FlexX (Bisson ct al. 2003
submitted). Connolly Surface was performed with Sybyl6.8
by Tripos Inc., St. Louis, USA.
16
AIM OF THE PROJECT STUDIED IN THIS THESIS
Progress in understanding the structure, function, and distribution of central nervous
system (CNS) nicotinic receptors and their pharmacology has opened up new
possibilities for novel CNS therapeutics with nicotinic agents (10,15-18). Their
potential benefit in a variety of neurodegenerative diseases involving the CNS, has
energized research efforts to design and develop neuronal nicotinic acetylcholine
receptor (nAchR) subtype selective ligands (10,27).
CHAPTERS II-III
The alkaloid epibatidine (EPB) (Figure 4) originally isolated from the skin of an
Ecuadorian poison frog, is a very specific agonist for the nAChRs exhibiting the
highest known affinities (subnanomolar range) towards the central brain nAChR a4ß2
as well as towards the homomeric nAChR 0:7 (21-26). This opened new ways to
investigate the properties of the pharmacophores of ligands for these receptors and thus
also for the properties ofthe binding sites ofthese receptors.
Unfortunately a possible therapeutic use ofEPB is prevented by its very high toxicity.
(-)-Cytisine (Figure 15) is a natural alkaloid that occurs in a large number of plants of
the Leguminosae family, and is well known as the main toxic principle of the common
garden Laburnum. (-)-Cytisine has one of the highest affinities for central brain
nAChR a4ß2- It behaves as a full agonist towards homomeric 017 nAChRs but with
lower potency than towards heteromeric nAChRs (60,61).
O
Figure 15. (-)-Cytisine. Chemical structure of (-)-cytisine.
17
In the present work, analogues of EPB and cytisine have been synthesised by our
group and by Dr. Rouden (University of Caen, France), respectively.
The compounds were then investigated using in vitro biochemical binding experiments
and electrophysiological approaches.
The binding experiments were done using the competition assay with [ H]-cytisine
(a4ß2 nAChR) and [125I]-alpha-bungarotoxin (a7 nAChR) as radioligands.
Voltage clamp electrophysiology was performed on neuronal (ot4ß2 and a7) and
ganglionic (oi3ß4) nAChRs reconstituted in Xenopus oocytes.
These in vitro studies are useful to investigate the relationship between ligand
chemical structure and nAChR subtype affinity and biological response in order to
develop high affinity and subtype selective nicotinic ligands.
CHAPTERS 1V-V
Recently the crystalline structure of the acetylcholine-binding protein (AChBP),
isolated from snails, has been resolved (Figure 9). It is a soluble homopentamer protein
produced and stored in glia cells and released into the synaptic cleft in an
acetylcholine-dependent manner to modulate neurotransmission. AChBP binds known
nAChR agonists and competitive antagonists such as acetylcholine, nicotine, d-
tubocurarine and a-bungarotoxin (12). It reveals, in the binding site region, high
similarities at the sequence and tridimensional level with the extracellular N-terminal
domain of different nAChR subunits to be then used as a modeling template.
The homology-models of the extracellular domain of the rat and human neuronal
(a4)2(ß2)3 and (a7)5 and of the rat ganglionic (a3)2(ß4)3 nAChR subtypes were built,
energetically minimized and their stability was assessed using molecular dynamics.
EPB has one of the highest affinities for the neuronal a4ß2 subtype but at the same
time has potent agonist activity at the ganglionic-K.3 containing subtype. The activity at
these peripheral nAChRs is apparently responsible for side effects like marked
hypertension observed with the in vivo use of epibatidine. This precludes then
epibatidine therapeutic usefulness (26). Many toxic clinical side effects may originate
from the non selectivity of certain ligands toward specific subtype receptors.
A series of nicotinic ligands were docked into the binding cavity of the rat a4ß2 and
a3ß4 subtypes. The structure-activity relationship was investigated to understand the
selectivity of ligands for the acetylcholine binding site of the rat a4ß2 and a3ß4
18
subtypes. The resulting models indicate how to design specific and selective <x4ß2
nAChR subtype ligands overcoming toxicity due to interaction with ganglionic a3ß4
receptor.
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24
CHAPTER 2
Synthesis and binding studies evaluation of epibatidine analogues as
novel ligands for the Nicotinic Acetylcholine Receptors
submitted to The Journal ofMedicinal Chemistry
Linjing Mu, William H. Bisson, Gerrit Westera*, Anita Schibig, Christa Wirz,
P.August Schubiger
Center for Radiopharmaceutical Sciences, Swiss Institute of Technology Zurich, Paul
Scherrer Institute, Villigen and University Hospital of Zürich, Rämistrasse 100,CH-
8091 Zürich, Switzerland
ABSTRACT
Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels
(LGIC), which are involved in a variety of functions like neurotransmission,
neurotransmitter release and control of cerebral blood flow. Recent research
demonstrated that selective nAChR ligands may have therapeutic potential in a number
of CNS diseases and disorders, including Alzheimer's disease (AD), nicotine
addiction, schizophrenia, and anxiety disorders. The alkaloid epibatidine [(-)-l] is a
highly potent non-opioid analgesic and nAChR agonist, but too toxic to be a useful
therapeutic or even diagnostic agent. To develop ligands selective for distinct nAChR
subtypes, reduced toxicity, and a satisfactory safety profile, a series of new epibatidine
analogues, compounds (±)-8, (±)-16, 18, 20, 21, (±)-22 were synthesized. The in vitro
binding affinities of these compounds showed significant differences towards the
nAChR subtypes a4ß2 and al. Compound (±)-22 demonstrated high affinity towards
a4ß2 (Kj = 2 nM), subtype selectivity (a4ß2 / al affinity ratio >100) and relatively
low toxicity on mice (LD50 > 0.5 mg/kg body weight) and will be labeled with nC and
18F to develop a new series of positron emission tomography (PET) tracers for imaging
nAChRs.
* Author for correspondence:Tel: xx41 1 255-35-60, Fax: xx41 1 255-44-28, ernail:[email protected]
25
Introduction
In the last ten years the rapid increase of knowledge about the structure,
function, and distribution of central nervous system (CNS) nicotinic receptors has
indicated the possibility for new CNS therapeutics with nicotinic ligands (1,2). In
particular, their potential in different neurodegenerative diseases involving the CNS,
has stimulated research efforts to develop neuronal nicotinic acetylcholine receptor
(nAChR) subtype selective ligands (3,4).
Nicotinic acetylcholine receptors (nAChRs) are excitatory ligand-gated cation
channels and are localized mostly in the central and peripheral nervous systems and
neuromuscular junctions. Moreover they mediate cholinergic neurotransmisson and
they are involved in important cognitive processes like learning and memory (5,6).
Neuronal nAChRs consist of a choice out of eight known a subunits (cc2 - ct9) and
three ß subunits (ß2 - ß4) in heterologous or homolougous pentamers (7,8), thus
building a variety ofnAChR subtypes.
The alkaloid epibatidine [(-)-l], isolated from the skin ofthe ecuadorian poison
frog Epipedobates tricolor by Daly and his coworkers (9) is a very specific agonist for
the nAChRs (9,10). It exhibites the highest known affinities (subnanomolar range)
towards the neuronal heteromeric a4ß2 (11) and homomeric al nAChRs (12).
Unfortunately, the use of epibatidine as a therapeutic agent is precluded by its very
high toxicity (13,14).
4
(-)-l (Epibatidine)
Despite of this, its binding affinity and agonistic activity opened new
possibilities to study the binding site ofthese receptors.
Analogues of epibatidine can be synthesized (15-18) and investigated using
biochemical (in vivo binding experiments) and electrophysiological approaches (19-
23) showing subtype-dependency affinities and activities.
26
Positron emission tomography (PET) is a non invasive technique to measure positron-
emitting radioisotopes for in vivo imaging. Hence, a variety of compounds labelled
with carbon-11 and fluorine-18 have been developed with potential as diagnostic PET
ligands.
In the last few years a certain number of ligands with high affinity for the ct4ß2
subtype have been identified. Epibatidine (24,25) and derivatives (15,26,27), cytisine
and derivatives (28,29) and 3-pyridyl ether compounds (30-32) have been radiolabeled
and tested for studying the nAChRs. However the clinical use of those radiotracers has
been limited due to toxicity problems (epibatidine analogues and derivatives) or due to
lipophilic labeled metabolites which can cross the blood brain barrier (3-pyridyl ether
compounds) (33).
In vivo imaging of nAChRs requires future development of new radioligands
with high binding affinity, appropriate lipophilicity (high extraction fraction), high
subtype selectivity (low toxicity), metabolism and blood clearance and appropriate
kinetics (34).
In the present work we synthesized a new series of epibatidine derivatives, we
determined in vitro binding affinities for cc4ß2 and al nAChRs, and we examined
structure-binding affinity relationships.
Results and Discussion
Chemistry. The synthesis of compound (±)-8 is shown on Scheme 1. First the
7-azabicycloheptadiene (±)-4 was constructed by the Diels-Alder reaction from the
Boc-protected pyrrole [2] with ethynyl sulfone 3 (35,36). The cycloadduct (±)-4 was
partially hydrogenated (Pd/H2) to give the vinyl sulfone intermediate (±)-5 in 93%
yield (37). By Michael type addition of 3-hydroxypyridine to compound (±)-5 the
pyridine moiety was introduced into this bicycloheptene system to afford a mixture of
the diastereoisomeric adducts (±)-6a and (±)-6b (38). The exo-[(±)-6a] and endo-[{±)-
6b] products were separated by column chromatography into nearly equal amounts.
Compound exo-(±)-6a was desulfonylated with 5% sodium amalgam to give
compound (±)-7 in 15% yield (35,39). The cleavage of the pyridine ether bond under
this condition makes it difficult to improve the yield. Other desulfonylation methods
like electrolysis (40) and the use of Samarium (II) iodide as a reductive agent (40,41)
27
did not give better results. After removing the Boc protecting group with TFA,
compound (±)-8 was obtained in 51% yield.
t-Boc
o 80 °C, 2 daysN +
Boc S02Tol78%
2 3
t-Boc
H2 (Pd/C), MeCIM
93%
S02Tol S02Tol
3-hydroxypyridine
NaH / DMF *
*o/
N
t-Boc /
40%
t-Boc
N
TFA, CH2C12
81% wS02Tol
\ Na/Hg, NaH2P04/Na2HP04\ I 6a
EtOAc / t-BuOH 6b
15%
Scheme 1. Preparation of (±)-2-(Pyridin-3-yloxy)-7-azabicyclo[2.2.1]heptane (8).
>Boc ,t-Boc .t-Boc
OBr
N +
I
Boc
90°C
46%
COOMe
Br
1) Et2NH,Et3N N
2)10% HCl
80%
COOMe
O Hj, Pd/C
COOMe
% ^~~~\10 11 12
1) 10% HCl
2) (Boc)20
COOMe
73%
Ht-Boct-Boc
.t-Boc .t-Boc
N Adam's catalyst N
86%
16 64%15 13
(TL ' 75*!N Br
14a
14b
Scheme 2. Preparation of (+)-2-(Pyridin-2-yloxy>7-azabicyclo[2.2.1]heptane (16).
The synthetic route for compound (±)-16 is illustrated in Scheme 2. Compound
(±)-10 was prepared starting from the commercially available Boc-protected pyrrole 2
and reacted with methyl bromopropiolate [9] which was obtained by bromination of 3-
methylpropiolate in the presence of AgNÛ3 as a catalyst (36,42). Compound 10 was
converted into the ß-ketoester (±)-ll by using a previously reported method (36). The
Diels-Alder adduct (±)-10 was treated with diethylamine in the presence of
28
triethylamine in acetonitrile, followed by hydrolysis in presence of 10% HCl to give
compound (±)-ll. After hydrogénation (10% Pd/C, H2) compound (±)-12 was
obtained in 98% yield. The ketone (±)-13 was prepared by decarboxylation of (±)-12
in presence of 10% HCl, then the free amine NH was protected again with di-tert-
butyl-dicarbonate (43). The ketone group was reduced in the presence of Adam's
catalyst to give two diastereoisomeric alcohols (±)-14a and (±)-14b in nearly 1:1 ratio,
(44) which were separated by column chromatography. Compound (±)-14a (exo-) was
then coupled with 2-bromopyridine in the presence of base (NaH), (S)-(-)-2,2'-bis(di-
p-tolylphosphino-l,l'binaphthyl (S-Tol-BINAP) and the
tris(dibenzylideneacetone)dipalladium chloroform complex (Pd2(dba)3 as catalyst, to
give compound (±)-15 in 75% yield. After deprotection by TFA (±)-16 was obtained in
64% yield.
As shown in Scheme 3, starting from commercially available tropine hydrate
17 (endo-), compound 18 was prepared with a reversed (exo-) configuration by the
Mitsunobu reaction with 3-hydroxylpyridine (45).
The synthetic route for compound 20 is shown in Scheme 4. 3-Bromopyridine was
lithiated with n-BuLi at -78°C then treated with tropinone [19] to present the tertiary
alcohol 20 in 64% yield (36).
The fluorinated product 21 was obtained by the reaction of compound 20 with
diethylamino sulfurtrifluride (DAST) (15% yield) together with the elimination
product (±)-22 (46% yield) (46-48).
T'11 DEAD,Ph3P ^—Y \, J17 OH 18 h
Scheme 3. Preparation of 8-Methyl-3-(pyridin-3-yloxy)-8-aza-bicyclo[3,2,1] octane (18).
29
Et2N-SF3——a
CH2C12
OH
22 (46%)
Scheme 4. Preparation of 3-Fluoro-8-methyl-3-pyridin-3-yl-8-aza-bicyclo[3,2,l]
(21) and (±)-8-methyl-3-pyridin-3-yl-8-aza-bicyclo [3,2,1] oct-2-ene (22).
The exo-endo isomerism in all cases was identified by 2D NMR with the
exception of compound 21 which was characterized by 'H-1D-NMR and MS due to
the low available amount.
In vitro binding studies
The inhibition constants (/Q of the novel ligands (±)-8, (±)-16, 18, 20, 21, (±)-
22 towards the cc4ß2 and al neuronal nicotinic receptors were estimated using the
inhibition competition binding assay method respectively with [3H]-cytisine (28) and
[125I]-a-bungarotoxin (49) (Table 1). The binding studies on the racemates (±)-8, (±)-
16, (±)-22 were performed without previous enantiomeric separation to have a first
idea of the binding affinity toward the nicotinic receptors.
All compounds showed low affinity towards the al neuronal nicotinic receptor,
which indicates high selectivity to a4ß2 subtype.
The affinity for the a4ß2 neuronal nicotinic receptor is related to the chemical
structure of the ligand. The identity and localization of three geometrically
independent key structural features for binding has lately been confirmed by
calculations using DISCO (Distance Comparison), QXP (Quick Explore) and MIPSIM
(Molecular Interaction Potential Similarity) software (50): (i) a positively charged
nitrogen atom for ionic or hydrogen bond interactions, (ii) a lone pair of the pyridine
30
Table 1. Binding affinities towards rat neuronal a4ß2 and a7 nAChRs
Ki ± SE,a nM
mpd [3H]-cytisine [ l]-a-bungarotc
(+)-l 0.053 ± 0.009 16±3
(-)-l 0.073 ± 0.01 17±3
(+)-2 0.064 ± 0.04b
(-)-2 0.015 ±0.03b
(±)-8 17.4 ±3.2 1290 ±47
(±)-16 620.5 ±271.8 2460 ± 369
18 955 ± 86 17800 ±2910
20 >20000 >20000
21 38.8 ±6.4 486 ±184
(±)-22 2.07 ±0.61 327 ±144
a
See Experimental Section. Data are presented as mean ± standard deviation.bIC50 data
nitrogen or a specific lone pair of carbonyl oxygen, as a hydrogen bond acceptor,
and (iii) a centre of a hydrophobic area generally occupied by aliphatic cycles.
The high inhibition constant of compound 20 (K\ > 20000 nM) is apparently due to
the presence of the OH group at C3 since the substitution of the OH group with
fluorine (compound 21) increased dramatically the affinity toward the a4ß2 receptor
(Ki - 38.8 nM).
Compound (±)-8 shows a much higher inhibition constant (K = 17.4 nM) than both
enantiomers of epibatidine [1] (K[(+) = 0.053 nM, K\(-) = 0.073 nM) and this means
that the insertion of an oxygen bridge between the azabicyclo and pyridine ring
decreased the affinity for the receptor and this may be due to the increase of the
distance between the quaternary and pyridine nitrogen atoms. The interatomic distance
and angles between the ligand's pharmacophoric groups have been proven to be
important for the affinity of the ligand to the receptor binding site. There seems to be
an optimal narrow range of distances. When the inter-nitrogen distance is increased by
31
changing the aza-heptabycyclo ring of compound (±)-8 to the aza-octabycyclo ring of
compound 18 the affinity decreases (50-fold, Ki = 955 nM), and when the N-N
distance is decreased by shifting the pyridine nitrogen from the 3- [(±)-8] to the 2-
position [(±)-16] the affinity also decreases (35-fold, K = 620 nM).
Compound (±)-22 shows high affinity (K\ ~ 2nM). The presence of a double bond
in the structure which limits the conformational flexibility increases the affinity for the
oc4ß2 receptor compared to the other compounds. Thus this space constrain would
establish more stable ligand-receptor interaction.
Among the racemates investigated only (±)-22 showed suitable affinity towards the
cc4ß2 nAChR subtype to be used as an in vivo PET tracer. The enantiomeric
separation of (±)-22 was avoided due to the high structural analogy between the
enantiomers. This analogy should give similar binding affinities to the brain receptor
investigated as demonstrated by Smith et al. (51).
In conclusion the affinity toward cc4ß2 subtype obtained with both enantiomers of
dechloroepibatidine [2] (15) (IC50(+) = 0.064 nM, IC50(~) = 0.015 nM) confirmed what
was found by Carroll et al. (35). The replacement of the 2'-chlorine of epibatidine [1]
with an hydrogen [2] or with another halogen atom (F, Br, I) doesn't play a role in the
affinity to the receptor. On the other hand, the 2'-substitution of epibatidine [1] with an
electron-withdrawing or electron-donating group seems respectively to increase or
decrease the affinity. Thus, electron-donating groups like hydroxy- and amino-groups
would somehow interfere negatively with the hydrogen-bond interactions between the
pharmacophore nitrogen pyridine and the receptor. Despite of this, no correlation have
yet been demonstrated between binding data and in vivo efficacy (17).
Experimental Section
Chemistry. All commercially available materials were used without further
purification unless otherwise stated. Solvents used for spectral measurement were of
spectrograde. Preparative separations were performed with flash column
chromatography (Merck silica gel 60, particle size 40-63 urn) and analytical TLC was
performed on precoated plates (Merck silica gel 60 F257); compounds were visualized
by using UV light or -I2 vapor. The proton and carbon NMR spectra were obtained on
32
a Bruker AMX-300 (300MHz) and 500MHz Spectrometer. Chloroform-d was used as
the solvent; Me4Si (5 0.00) was used as an internal standard. All NMR chemical shifts
are reported as 3 values and coupling constants (J) are given in hertz (Hz). The
splitting pattern abbreviations are as follows; s, singlet; d, doublet; t, triplet; q, quartet;
m, multiplet; br, broad. Melting point is defined as mp. The electrospray mass spectra
were carried out using a Finnigan Mat Instrument TSQ 7000.
(±)-7-/er/-Butoxycarbonyl-2-/»-tolylsulfonyI-7-azabicyclo[2.2.1]hepta-2,5-diene
(4). A stirred solution of ethynyl p-tolyl sulfone (1.8 g, 0.01 mol) in N-tert-
butoxycarbonylpyrrole (3.34g, 0.02 mol) was heated to 80 °C under argon and the
reaction mixture was protected from light. After 48 h, the obtained black oil was
cooled down to room temperature and purified by column chromatography (silica gel,
EtOAc/hexane = 1:5 —> 1:3) to yield recovered N-/er/-butoxycarbonylpyrrole (2.88 g)
followed by the cycloadduct as a pale yellow solid (2.36 g, 68%). Mp 97-98 °C. !H
NMR (CDC13): Ô 7.75(d, J = 8.32 Hz, 2H), 7.56( br. 1H), 7.35( d, J = 8.08 Hz, 2H),
6.95( br, 1H), 6.87(dd, J = 5.04, 2.52 Hz, 1H), 5.38(br, 1H), 5.17(s,lH), 2.45(s, 3H),
1.27(br, s, 9H); 13C NMR (CDCI3): Ô 158.9 (C=0), 154.2 (C ), 152.6 (CH), 145.3 (C ),
143.0 (CH), 141.9 (CH), 136.1 (C ), 130.4 (CH), 128.5 (CH), 81.8 (CMe3), 68.1 (CH),
67.2 (CH), 28.2 (CM?5), 22.0 (CH3).
(±)-7-*tv-/-Butoxycarbonyl-2-p-tolyIsulfonyl-7-azabicyclo[2.2.1]hepta-2-ene
(5).The diene (±)-4 (1.06 g, 3.05 mmol) was dissolved in acetonitrile (15mL) and Pd/C
(0.1 g, 10%) was added in one portion. The suspension was stirred under H2 until the
required volume of H2 was absorbed (68 mL, 3.05 mmol). The reaction mixture was
filtered through celite and the filtrate concentrated in vacuo. The residue was
chromatographed on silica gel (EtOAc/hexane = 1:4) to yield the title compound as a
white solid (1 g, 93%). Mp 97-98 °C. 'H NMR (CDCI3): 8 7.80 (d, J = 8.1 Hz, 2H),
7.35 (d, J = 8.0 Hz, 2H), 7.05 (d, J = 1.9 Hz, 1H), 4.83 ( br, 1H), 4.76 (d, J = 3.5 Hz,
1H), 2.45 (s, 3H), 1.98 (m, 2H), 1.38 (m, 1H), 1.21 (br, 10H, -COz'Bu+Ç-H); MS m/z
(ESI) 372.1 [M + Na]+.
(±)-7-ter/-ButoxycarbonyI-2(pyridin-3-yloxy)-3-p-tolylsulfonyl-7-azabicycIo-
[2.2.1]heptane (6). To a stirred solution of 3-hydroxypyridine (98 mg, 1.02 mmol) in
dry DMF (7 mL), NaH (24 mg, 1.02 mmol) was added in one portion. After 10 min.
stirring at room temperature, (±)-5 (300 mg in 2 mL dry DMF, 0.86 mmol)) was added
33
drop-wise, then the reaction solution was stirred at room temperature for 24 h. The
solvent was removed by high vacuum, the residue was partitioned between CH2CI2 and
saturated NH4CI in H2O. The aqueous phase was extracted with CH2CI2 three times,
the combined organic layers were dried over MgS04, filtered and concentrated. The
crude product was purified by column chromatography (silica gel
EtOAc/DCM/hexane, 3:3:4) and provided two oil isomeric products (±)-6a and (±)-6b
in nearly equal amounts (yield 40%). !H NMR (endo CDCI3): Ö 8.25 (d, J = 4.28 Hz,
1H), 8.10 ( br, 1H), 7.73 (dd, J = 6.58, 1.78 Hz, 2H), 7.28 (m, 2H), 7.18 (m, 1H), 7.07
(m, 1H), 4.74 ( br, 1H), 4.58 (t, J = 4.28 Hz, 1H), 4.40 (d, J = 5.56 Hz, 1H), 3.75 ( br,
1H), 2.40 (s, 3H), 1.99 ( m, 1H), 1.75 (m, 1H), 1.66 ( br, 2H), 1.33 ( br, 9H);13C NMR
(CDC13): 5 153.6, 145.6, 143.8, 137.1, 130.5, 128.2, 124.2, 82.9, 81.2, 28.5, 22.0; MS
m/z (ESI) 445.3 [M + 1] +; !H NMR (exo CDC13): ö 8.26 (d, J = 4.04 Hz, 1H), 8.19 (
br, 1H), 7.75 (d, J = 7.6, 2H), 7.28-7.21 (m, 4H), 4.91 ( br, 1H), 4.61 (d, J = 4.8 Hz,
1H), 4.52 ( br, 1H), 3.19 (d, J = 4.04 Hz, 1H), 2.39 (s, 3H), 1.90 (m, 2H), 1.63 ( br,
2H), 1.45 (s, 9H);13C NMR (CDC13): S 154.0, 153.9, 145.5, 144.0, 139.9, 134.9,
130.3, 129.3, 124.3, 81.2, 78.8, 73.6, 58.1, 28.6, 22.0; MS m/z (ESI) 445.2 [M + 1]+.
(±)-7-fer/-Butoxycarbonyl-2(pyridin-3-yloxy)-7-azabicycIo[2.2.1]heptane (7). To
a stirred mixture of Na2HP04 (255 mg, 1.8 mmol) and NaH2P04 (216 mg, 1.8 mmol)
in 5 mL of 1:1 EtOAc and tert-buXy\ alcohol, 5% Na/Hg (3.3 g, 7.2 mmol) was added
at 0°C under argon, (±)-6a (160mg in 2mL 1:1 EtOAc and /erf-butyl alcohol, 0.36
mmol) was added drop-wise to the reaction mixture. After 1 h stirring at 0°C and 3 h at
room temperature, the solid was removed by filtration, washed with CH2CI2 and
EtOAc, the organic solvents were removed at reduced pressure. The residue was
partitioned between H2O and CH2CI2, the aqueous phase was extracted two times with
CH2CI2, the organic layers combined, dried over Na2S04, filtered and evaporated. The
crude product was purified by column chromatography on silica gel
(EtOAc/MeOH/hexane, 3:1:6) to give 16 mg of 7 in the form of oil (yield 15%).!H
NMR (CDCI3): 8 8.36 (d, J = 2.76 Hz, 1H), 8.24 (m, 1H), 7.20 (m, 1H), 7.16 (m, 1H),
4.98 (d, J = 9.6 Hz, 1H), 4.45 (s, 1H), 4.24 (s, 1H), 2.35 (m, 1H), 2.13 (m, 1H), 1.84
(m, 1H), 1.66 (m, 2H), 1.56 (m, 1H), 1.46 (s, 9H); MS m/z (ESI) 291.3 [M + 1]+.
(±)-2-(Pyridin-3-yloxy)-7-azabicyclo[2.2.1]heptane (8). To a solution of the
compound (±)-7 (15 mg) in CH2CI2 (0.5 mL) was added trifluoroacetic acid (TFA, 0.5
34
mL). The resultant solution was stirred at room temperature for 1 h. Evaporation of
both solvent and TFA at reduced pressure, the trace of remaining TFA was removed by
repeated evaporation with CH2CI2 and MeOH. The residue was purified by column
chromatography (silica gel, CH2Cl2/MeOH/Et3N, 10:1:0.11) providing a yellowish oil
of the desired product (±)-8 (5mg, 51%). !H NMR (CDCI3): 8 8.28 (d, J - 2.3 Hz, 1H),
8.20 (d, J = 3.6 Hz, 1H), 7.18 (m, 2H), 4.67 (m, 1H), 3.92 (t, J = 4.5 Hz, 1H), 3.73 (t, J
= 4.7 Hz, 1H), 2.25-1.51 (m, 7H); MS m/z (ESI) 191.0 [M + 1]+.
Methyl 3-bromopropioIate (9). To a solution of methyl propiolate (2 g, 23.8 mmol)
in 75 mL acetone at room temperature, the catalyst silver nitrate (AgN03, 0.4 g, 2.35
mmol) was added, followed by N-bromosuccinimide (NBS, 4.9 g, 27.5 mmol) all in
one portion. The homogeneous solution was stirred at room temperature and turned
cloudy within ca. 5 min, then a grayish precipitate developed. After 1 h stirring, the
solvent was evaporated, then distilled under high vacuum. The compound was
collected in a cooling trap bottle (3.5 g, 90%).
(±)-Methyl-2-bromo-7-(/ert-butoxycarbonyl)-7-azabicyclo[2.2.1]hepta-2,5-diene-
2-carboxyIate (10). A mixture of methyl 3-bromopropiolate (3.5 g, 21 mmol) and N-
Boc-pyrrole (17.5 g, 105 mmol) was stirred at 90°C under argon for 30 h. After
cooling down to room temperature, the resulting mixture was subjected to column
chromatography (silica gel, EtOAc/petroleum ether, 1:15). The first fraction contained
non reacted N-Boc-pyrrole (13 g), followed by the title compound (±)-10 (3.2 g, 46%)
as a slightly yellow oil/H NMR (CDC13): ô 7.12 ( br, s, 2H), 5.48 (s, 1H), 5.13 (s, 1H),
3.79 (s, 3H), 1.41 (s, 9H).
(±)-7-(/eri,-Butoxycarbonyl)-3-(methoxycarbonyl)-7-azabicyclo[2.2.1Jhept-5-ne-
2-one (11). (±)-10 (2.6 g, 7.8 mmol) and triethylamine (Et3N, 5.5 ml, 39 mmol) were
dissolved in 20 mL of acetonitrile under argon, a solution of diethylamine (0.9ml,
8.5mmol) in acetonitrile (13ml) was added drop-wise to the above solution. After 1.5 h
stirring at room temperature, a 10% HCl (26 mL) solution was then added drop-wise to
the reaction mixture at ca. 5°C. The reaction mixture was stirred at room temperature
for an additional 4 h. A portion of H2O (26 mL) was added, the mixture was extracted
with CH2CI2 (3 x 35 mL). The organic phases were combined, dried over Na2S04,
filtered and concentrated under reduced pressure. The residue was purified by column
35
chromatography (silica gel, EtOAc/petroleum ether, 1:5) to give the desired compound
(±)-ll (1.68g, 80%) as an oil (endo/exo, 7:1).'H NMR (CDC13): S 6.95
( br, s, 2H), 4.8-4.7 (m, 1H), 4.3 (m, 1H), 3.73 (s, 3H), 2.5 (m, 1H), 1.41 (s, 9H).
(±)-7-(*'e*r/-Butoxycarbonyl)-3-(methoxycarbonyl)-7-azabicyclo[2.2.1]heptan-5-
ne-2-one (12). To a solution of (±)-ll (1.1 g, 4.1 mmol) in MeOH (15 mL), Pd/C (110
mg, 10%) was added under argon. The reaction mixture was hydrogenated with
vigorously stirring at latm and room temperature for 10 h. After finishing, the catalyst
was removed by filtration, and the filtrate was concentrated under reduced pressure.
The residue was purified by flash column chromatography (silica gel,
EtOAc/petroleum ether: 1:5) to provide the ß-keto ester (±)-12 in a form of oil (yield
98%, endo/exo = 3:2).
'H NMR (CDCI3): 8 4.83 (d, J = 3.7 Hz, 0.4H), 4.72 (t, J = 4.4 Hz, 0.6H), 4.35 (d, J =
3.7 Hz, 0.4H), 4.32 (d, J = 4.4 Hz, 0.6H), 3.74 (s, 1.2H), 3.72 (s, 1.8H), 3.44 (d, J = 5.0
Hz, 0.6H), 2.99 (s, 0.4H), 2.05 (m, 2H), 1.82 (m, 2H), 1.45 (s, 9H).
(±)-7-(fer/-Butoxycarbonyl)- 7-azabicyclo[2.2.1]heptan -2-one (13). A mixture of
ß-keto ester (±)-12 (0.87 g, 3.2 mmol) and 10% HCl (78 mL) was heated at 100-110°C
oil bath for 3h under argon. After cooling down to room temperature, the solvent was
evaporated under reduced pressure; the remaining trace of water was removed by
azeotropic distillation with EtOH and dried by high vacuum. The residue was
dissolved in dry CH2C12 (50 mL), then triethylamine (Et3N, 1.77 mL, 12.8 mmol) and
di-te/?-butyl-dicarbonate ((Boc^O, 1.4 g, 6.4 mmol) were added. The solution was
stirred at room temperature overnight. After finishing, the reaction mixture washed
with saturated Na2C03, the aqueous phase was extracted with CH2CI2 (3 x 30mL), the
combined organic layers were dried over Na2S04, filtered and concentrated. The
yellow oil residue was purified by chromatography (silica gel, EtOAc/petroleum ether:
1:5) to give the ketone (±)-13 (500mg, 73% yield) as a colorless oil. ]H NMR (CDCI3):
8 4.55 (t, J = 4.6 Hz, 1H), 4.24 (d, J = 5.2 Hz, 1H), 2.47 (dd, J = 17.5, 5.2 Hz, 1H),
1.99-2.04 (m, 3H), 1.55-1.68 (m, 2H), 1.45 (s, 9H); MS m/z (ESI) 212.0 [M + 1]+.
(±)-7-(fer,,-Butoxycarbonyl)- 7-azabicyclo[2.2.1]heptan -2-ol (14). A suspension
of (+)-(+)-!3 (210 mg, 0.99 mmol) and Adam's catalyst (Pt02, 40 mg, 0.17 mmol) in
anhydrous methanol (5 ml) was vigorously stirred at room temperature under H2 at 1
atm for 24 h. After finishing, the catalyst was removed by filtration, and the filtrate
36
was concentrated under reduced pressure. The residue was purified by flash column
chromatography (silica gel, EtOAc/petroleum ether: 1:2) to afford the 7-Boc-7-
azaicyclo[2.2.1]heptan-2-ols (yield 86%, exo((±)-14a)/endo ((±)-14b), 1:1). Mp (exo)
68-70 °C. lK NMR (endoCDCh): 6 4.34 (m, 1H), 4.12 ( br, 2H), 2.15(m, 2H), 1.96 (m,
1H), 1.43 (s, 9H), 1.81-1.30 (m, 4H); MS m/z (ESI) 449.2 [2M + Na] +;*H NMR (exo
CDC13): 8 4.21 ( br, 1H), 4.10 ( br, 1H), 3.84(m, 1H), 2.05-1.39 (m, 7H), 1.43 (s, 9H);
MS m/z (ESI) 449.2 [2M + Na]+.
(+)-7-*V/-/,-Butoxycarbonyl-2(pyridin-2-yIoxy)-7-azabicyclol2.2.1]heptane (15).
To a solution of (±)-14a (97 mg, 0.45 mmol) in xylene (9 mL), sodium hydride (95%
NaH, 23 mg, 0.91 mmol) was added under argon, the mixture was stirred at 70°C for
25 min. After cooling down to room temperature, 2-bromopyridine (72 mg, 0.45
mmol), tris(dibenzylideneacetone)dipalladium chloroform complex (Pd2(dba)3, 15 mg,
6% Pd,) and (S)-(-)-2,2'-bis(di-p-tolylphosphino)-l,l'-binaphthyl (Tol-BINAP, 23 mg,
0.03 mmol) were added to the above solution, the reaction mixture was then stirred on
a 90°C oil bath for 20 h. After removal of the solvent on an evaporator, the residue was
purified by column chromatography (silica gel, Hexane/EtOAc, 4:1) to afford an oil of
the title compound (±)-15 (100 mg, 75%). !H NMR (CDCI3): 8 8.12 (d, J = 3.6 Hz,
1H), 7.54 (t, J = 7.2 Hz, 1H), 6.84 (t, J = 5.9 Hz, 1H), 6.69 (d, J = 8.3 Hz, 1H), 4.98
(dd, J = 6.7, 2.6 Hz, 1H), 4.41 (d, J = 4.9 Hz, 1H), 4.31 ( br., 1H), 1.92 (m, 1H), 1.39
(s, 9H), 1.49-1.23 (m, 5H);13C-NMR (CDC13): S 163.2, 156.1, 146.9, 138.6, 116.8,
112.0, 79.5, 60.4, 55.1, 39.3, 28.4, 24.7; MS m/z (ESI) 313.1 [M + Na]+.
(±)-2-(Pyridin-2-yloxy)-7-azabicyclo[2.2.1]heptane (16). To a solution of the
compound (±)-15 (95 mg, 0.32 mmol) in CH2C12 (2.5 mL) was added Trifluoroacetic
acid (TFA, 1 mL). The reaction mixture was stirred at room temperature for 1 h. After
evaporation of both the solvent and TFA at reduced pressure, the trace of remaining
TFA was removed by repeated evaporation with CH2C12 and MeOH. The residue was
purified by column chromatography (silica gel, EtOAc/hexane/Et3N, 1:1:0.02)
provided the desired product (±)-16 in the form of oil (40 mg, 64%).]H NMR (CDCI3):
S 8.13 (d, J = 3.8 Hz, 1H), 7.55 (t, J = 8.2 Hz, 1H), 6.84 (t, J = 5.8 Hz, 1H), 6.69 (d, J =
8.3 Hz, 1H), 4.99 (t, J = 5.7 Hz, 1H), 3.71 (m, 2H), 2.00 (m, 1H), 1.65 (m, 3H), 1.27
(m, 3H);13CNMR (CDCI3): 8 162.9, 146.7, 138.4, 116.5, 111.5, 78.1, 60.6, 55.3, 41.0,
28.8, 24.3; MS m/z (ESI) 191.1 [M + 1]+.
37
8-Methyl-3-(pyridin-3-yloxy)-8-aza-bicyclo [3,2,11 octane (18). Tropine hydrate,
17 (0.7 g, 4.96 mmol) was dissolved in 15 mL dry THF, then triphenylphosphine
(1.56 g, 5.95 mmol) and 3-hydroxypyridine (0.57 g, 5.95 mmol) were added. The
solution was cooled down to 0°C and diethylazodicarboxylate (DEAD, 1.04 g in 5 ml
dry THF, 5.95 mmol) was added drop-wise. After 10 min, the ice bath was removed
and the reaction mixture was stirred at room temperature for 20 h. After evaporation
of the solvent, the residue was dissolved in methylene chloride and washed with IN
HCl. The aqueous layer was adjusted to basic pH with aqueous Na2C03 and extracted
with methylene chloride. The organic extract was dried over Na2S04, filtered and
concentrated under reduced pressure. The residue was purified by column
chromatography (silica gel, CH2Cl2/Me0H, 9:1) provided the white solid product 18
(yield 50%). Mp 160 °C. lH NMR (CDC13): 8 8.27 (s, 1H); 8.17 (m, 1H), 7.17 (m,
2H), 4.51 ( m, 1H), 3.26 ( m, 2H), 2.36 (s, 3H), 2.10 ( m, 2H), 1.99 ( m, 2H), 1.94 ( m,
2H), 1.62 ( m, 2H);13C NMR (CDCI3): 8 154.38, 142.56, 139.71, 124.19, 123.28,
71.32, 61.12, 38.69, 36.09, 27.38; MS m/z (ESI) 219.0 [M + 1]+.
8-M ethyl-3-pyridin-3yl-8-aza-bicyclo[3,2,l] octan-3-ol (20). 3-bromo-pyridine (
0.95 g, 0.6 mmol) was dissolved in 20ml absolute Et20, then cooled down to -78°C, n-
BuLi ( 1.6 M in Hexane, 2.5 mL, 0.39 mmol) was added to the solution dropwise, the
reaction mixture was stirred at -78°C for 30min, and then 8-methyl-8-aza-bicyclo
[3,2,1] octan-3-one was added in one portion. After, the mixture was stirred at -78°C
for another 10 min, the cooling bath was removed, the mixture was stirred at room
temperature for 12 h. 5 mL H2O was added to quench the reaction, and the organic
phase was separated, aqueous phase was extracted with CH2CI2. The combined organic
layers were dried over K2CO3 and Na2S04, filtered and concentrated under reduced
pressure. The residue was purified by column chromatography (silica gel,
EtOAc/MeOH/Et3N, 2:1:0.03), which gave 20 (0.699 g, 64%) in the form of white
solid. Mp 83 °C. !H NMR (CDCI3): 8 8.77 (d, J = 2.60 Hz, 1H), 8.45 (dd, J = 4.6, 1.6
Hz, 1H), 7.80 (m, 1H), 7.21 (m, 1H), 3.26 (m, 2H), 2.35 (s, 3H), 2.41-2.32 (m, 2H),
2.21 (m, 2H), 2.05 (m, 3H), 1.87 (m, 1H), 1.80 (m, 1H); MS m/z (ESI) 219.2 [M + 1]+.
3-Fluoro-8-methyl~3-pyridin-3-yl-8-aza-bicycIo[3,2,1] (21) and (±)-8-methyl-3-
pyridin-3-yl-8-aza-bicyclo (3,2,1] oct-2-ene (22). To a solution of 20 (100 mg, 0.46
mmol) dissolved in dry CH2C12, diethylaminosulfurtrifluoride (DAST) (100 ul, 1.5 eq)
was added at 0°C, the mixture was stirred at 0°C for 2 h, then warmed up to room
38
temperature and stirred overnight under argon. To the reaction solution, H2O saturated
with NaHC03 was added, the two phases separated, the aqueous phase was extracted
with CH2CI2, the organic layers combined, dried over Na2S04, filtered and
concentrated. The residue was purified by column chromatography (silica gel, EtOAc
MeOH/Et3N 2:1:0.03), which gave 21 (15 mg, 15%) and (±)-22 (42 mg, 46%) in the
form of yellowish oils. lti NMR (21-CDC13): S 8.74 (d, J = 1.5 Hz, 1H), 8.50 (dd, J =
5.1, 1.5 Hz, 1H), 7.87 (d, J = 7.8 Hz, 1H), 7.24 (m, 1H), 3.47 (s, 2H), 2.52 (s + m, 3H
+ 2H), 2.14 (m, 6H); MS m/z (ESI) 221.1 [M + 1]+; JH NMR ((±)-22-CDCl3): 8 8.59
(d, J = 1.6 Hz, 1H), 8.42 (dd, J = 4.7, 1.5 Hz, 1H), 7.60 (m, 1H), 7.19 (m, 1H), 6.29 (d,
J = 5.5 Hz, 1H), 3.47 (m, 2H), 2.86 (dd, J = 17.2, 3.7 Hz, 1H), 2.41 (s, 3H), 2.15 (m,
3H), 1.88 (m, 1H), 1.61(m, 1H); MS m/z (ESI) 201.0 [M+ 1]+.
In Vitro Binding Studies. Materials. (-)-[3H] Cytisine (32.4 Ci/mmol) and [125I]-a-
Bungarotoxin (146 Ci/mmol) were obtained from PerkinElmer Life Sciences (Boston,
USA). (-)-Nicotine and a-bungarotoxin were obtained from Sigma-Aldrich
(Steinheim, Germany) and all buffer chemicals from Fluka (Buchs, Switzerland).
Frozen Sprague-Dawley rat brains were purchased from RCC Ltd (Füllinsdorf,
Switzerland) and Charles River Labs (Sulzfeld, Germany).
Competition Binding Assay. Binding assays of all the compounds synthesized for the
cc4ß2 and al neuronal nicotinic receptor subtypes were performed at the final volume
of 0.2 ml containing 100 ug of protein (membrane preparations of frozen Sprague-
Dawley rat brains) in a BSS buffer at pH 7.4. The concentration of [3H]-cytisine
(72000-59600 dpm/pmol) was 2 nM. After lh at 0°C, the samples were vacuum-
filtered on glass fiber filters (Whatman GF/C) which were then washed with ice cold
buffer (2x4 ml). Non-specific binding was estimated in the presence of 0.1 mM
nicotine. Packard Ultima Gold was the cocktail used for liquid scintillation counting
with a Tri-Carb 2200CA analyzer. The concentration of [125I]-alpha-bungarotoxin
(213839-102091 dpm/pmol) was 0.5 nM. After 2 h at 37°C, the samples were vacuum-
filtered on glass fiber filters (Whatman GF/B) which were then washed with ice cold
buffer (3x4 ml). Non-specific binding was estimated in the presence of 0.01 mM
alpha-bungarotoxin. The radioactivity was counted with a Packard Cobra II
autogamma.
39
Data Analysis. The binding results from the counter were analysed using the Kell
Programme from Biosoft using respectively Kd = InM for (-)-[3H] Cytisine (a4ß2
subtype) and Kd = 0.7 nM for [125I]-a-Bungarotoxin (al subtype).
Conclusions
(±)-Epibatidine [(±)-l] is a very specific agonist for the nAChRs. Both
enantiomers exhibit the highest known affinities (subnanomolar range) towards the
central brain heteromeric nAChR a4ß2 as well as towards the homomeric nAChR al.
In order to develop new ligands for the different existing subtypes of nAChRs with
high affinity, better subtype selectivity but lower toxicity, we have synthesized and
evaluated by binding studies some epibatidine analogues. These compounds have all
the key pharamacophore characteristics important for the binding to the nicotinic
receptor but the interatomic distances and angles between these structural features
change from ligand to ligand and this influences the receptor binding and as a
consequence the subtype selectivity. Beyond these key pharmacophore factors, the
direction of the hydrogen bond and other physicochemical features have to be
considered to account for structure-affinity relationships.
All compounds [(±)-8, (±)-16, 18, 20, 21, and (±)-22] show higher affinity
towards cc4ß2 compared to a7 nAChRs. The a4ß2 subtype affinity depends strongly
on subtle structural features of the ligand. Of this series of compounds (±)-22 shows
the highest affinity for the a4ß2 nAChRs subtype (Ai ~ 2nM) and relatively low
toxicity in mice. Thus, the N(8)-uCH3-analogue of compound (±)-22 could be useful
as a PET tracer for imaging the brain nAChRs.
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46
CHAPTER 3
Affinity and electrophysiological evaluation of epibatidine and
cytisine analogues as novel ligands for the Nicotinic
Acetylcholine Receptors
submitted to Chemistry and Biology
1William H. Bisson,h* Gerrit Westera,
2Sonia Bertrand,x Linjing Mu,1 Anita
Schibig,3 Jacques Rouden,2 Daniel Bertrand andlP. August Schubiger
1Center for Radiopharmaceutical Sciences, Swiss Institute ofTechnology Zurich, Paul
Scherrer Institute, Villigen and University Hospital, Zurich, Switzerland2Physiology Department, University Medical Centre (CMU), Geneva, Switzerland
3Laboratoriesfor Molecular and Thio-organic Chemistry, University ofCaen, Caen,
France
SUMMARY
Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand gated ion channels
(LGIC), which are involved post-synaptically in the neurotransmission and pre-
synaptically in the modulation of neurotransmitter release into the synaptic cleft.
Selective nAChR ligands may have therapeutic potential in a number of CNS diseases
and disorders. To develop new ligands selective for distinct nAChR subtypes with
potent analgesic activity, reduced toxicity, and a satisfactory safety profile, new
epibatidine analogues, compounds (±)-l, 2, 3, 4, (±)-5, (±)-6 and cytisine analogues,7-
8 have been synthesized. The in vitro binding affinities for the new series differentiate
significantly among the nAChR subtypes (a$2 and aj) investigated. Voltage clamp
electrophysiology was performed with some of these compounds on the neuronal 0^2
and a7 and on the ganglionic a3ß4 nAChRs reconstituted in Xenopus oocytes.
Compound,(±)-6, demonstrated high affinity and partial activity on a4ß2 (2 nM),
subtype selectivity and low toxicity on mice (LD50 > 0.5 mg/kg body weight) and will
be the starting point for development of nCH3 and 18F-labeled derivatives as positron
emission tomography (PET) tracers for imaging nAChRs.
* Author for correspondence:Tel: xx41 1 255-35-60, Fax: xx41 1 255-44-28, email:[email protected]
47
Introduction
Recent development to investigate the structure, biological role, distribution and
pharmacology of central nervous system (CNS) nicotinic receptors have raised
research interest in the therapeutic use of CNS nicotinic agents (1,2). They have been
proved to be useful in a variety of neurodegenerative diseases. As a consequence the
effort to develop neuronal nicotinic acetylcholine receptor (nAChR) subtype selective
ligands has increased (3,4).
Nicotinic acetylcholine receptors (nAChRs) are excitatory ligand-gated cation
channels localized mostly in the central and peripheral nervous systems and
neuromuscular junctions. They are involved in the cholinergic neurotransmission
mediation and nerve pulse conduction in brain cognitive areas for processes like
learning and memory (5,6). Neuronal nAChRs are heterologous or homologous
pentamers (7,8) composed of a (cc2 - ocq) and ß (ß2 - ß4) subunits. Thus this subunit
diversity permits a variety of nAChR subtypes with distinct cationic conduction and
pharmacological properties.
The alkaloid epibatidine, originally isolated from the skin of the Ecuadorian poison
frog Epipedobates tricolor (9), is a very specific agonist for the nAChRs (10) showing
the high affinities (subnanomolar range) towards the central brain nAChR a4ß2 and a7
(11,12). Unfortunately, the clinical therapeutic use of epibatidine is precluded by its
high toxicity (13-15). Although its binding and activity properties have been used as a
template for new investigations on the binding site of these receptors.
Cytisine is a natural alkaloid found in a large number of plants of the Leguminosae
family and known to be the main toxic component of the garden Laburnum. Binding
studies (16,17) and functional studies (18) have shown that cytisine has one of the
highest affinities for central brain a4ß2 nAChR. In addition, cytisine behaves as a full
agonist towards homomeric aj although in a less extent than towards heteromeric
nAChRs (19,20).
Therefore, analogues of epibatidine and cytisine have been synthesised (21-25) and
investigated using biochemical (in vitro binding experiments) and electrophysiological
approaches (15,26-30).
48
Positron emission tomography (PET) is an in vivo non-invasive imaging technique and
a variety of PET compounds have been labeled with carbon-11 and fluorine-18 to
develop diagnostic brain receptor tracers.
In the last few years researchers have identified different ligands with high affinity for
the a4ß2 subtype. In particular, epibatidine and derivatives (31-34), cytisine and
derivatives (16,25) and 3-pyridyl ether compounds (35-37) have been radiolabeled
and tested for studying nAChRs. However toxic side effects and problems due to
undesired metabolites (38) have affected nicotinic radioligands-research. Compounds
with high binding affinity, moderate lipophilicity, high subtype selectivity, optimal
rates of metabolism and blood clearance have to be achieved (39).
In the present work we synthesised new analogues of epibatidine and cytisine, we
measured in vitro binding affinities towards a4ß2 and a7 nAChRs and we examined
structure - binding affinity relationships for these compounds. Moreover, voltage-
clamp electrophysiology (40,41) was performed with some of these compounds on the
neuronal a4ß2 and ot7 and on the ganglionic oi3ß4 nAChRs to investigate agonist and
antagonist activity towards these receptors.
Results and Discussion
In vitro binding studies
In vitro inhibition constant (Kj) evaluation was performed using the inhibition binding
assay. We used radioligands [3H]-cytisine (16) and [125I]-a-bungarotoxin (42)
respectively towards cc4ß2 and a7 nAChR subtypes. The structure-affinity relationship
of the synthesised compounds (±)-l, 2, 3, 4, (±)-5, (±)-6, 7, 8 (Figure 1) was
investigated. The Ki values are shown in Table 1.
Preliminary binding studies were performed on the racemates (±)-l, (±)-5, (±)-6
without previous enantiomeric separation to obtain a first idea of the binding potential
toward the nicotinic receptors.
The binding affinity towards a7 nAChR subtype resulted to be low independently of
the chemical structure involved. Thus this demonstrates that epibatidine derivatives
(±)-l, 2, 3, 4, (±)-5, (±)-6 are oc4ß2 subtype selective. The highest affinity within the
series is obtained with the cytisine derivative, 8. The affinity of cytisine [10] (Kj = 260
nM) is dramatically reduced with the insertion of a p-fluoro-phenyl group at 3-
49
position (7, Ki > 20000 nM). On the other hand, the insertion of an halogen atom, like
bromine,increases the affinity, although, the binding potential remains not high (8, Ki
= 7.47 nM ).
A significant affinity-structure dependency towards a4ß2 nAChR subtype was found.
3-Bromocytisine (8) has a very low and 3-(4,-fluorophenyl)cytisine (7) a very high
inhibition constant: the insertion of a bromine atom in position 9 of cytisine [10] (K; ~
2 nM) increased the affinity 10 fold while the insertion at the same position of a 4'-
fluorophenyl group decreased the affinity by two orders of magnitude. This means that
the nature of the substituent at position 3 has a big impact on the pharmacophore, most
notably on the affinity toward the different receptor subtypes.
Table 1,Binding Data for Epibatidine, Cytisine and Analogues *
A, ± SE,a nM
îpd [3H]-cytisine [125I]-a-bungarotoxin
1 17.4 ±3.2 1290 ±47
2 955 ± 86 17800 ±2910
3 >20000 >20000
4 38.8 ±6.4 486 ±184
5 620.5 ±271.8 2460 ± 369
6 2.07 ±0.61 327 ±144
7 48.3 ±4.4 >20000
8 0.153 ±0.034 7.47 ± 2.53
+)-9 0.053 ± 0.009 16 ±3
-)-9 0.073 ± 0.01 17±3
-)-10 1.2±0.4a 260 ± 20b
aIC50(nM)fromref[16]bfromref[25]* Data are presented as mean ± standard deviation
50
H2N+
H2N+
H2N+
*Br
9 epibatidine 10 cytisine
Figure 1. Epibatidine and cytisine analogues. Epibatidine and cytisine
analogues synthesised.
51
Probably the phenyl group of the 3-(4'-fluorophenyl)cytisine (7) changes the ligand
orientation in the binding site of the receptor, with the subsequent loss of all binding
interactions which are active with a bromine (3-bromocytisine (8) ), chlorine or iodine
substituent (25). Imming et al. showed that, within the different halogen-derivatives,
the one with bromine reveals the highest affinity. This is probably due to the fact that
bromine has atomic properties like electronegativity and dimension which are suitable
for optimal binding.
Regarding the epibatidine derivatives (±)-l, 2, 3, 4, (±)-5, (±)-6 the impact of four
structural factors on affinity has been studied: the bulkiness of the central hydrophobic
area represented by the aliphatic cycle; the position of the hydrogen bond acceptor
nitrogen in the pyridinyl ring; the type of substituent in the aza-octabicyclo-derivatives
and the insertion of an oxygen in the bridge between the pyridine ring and the aliphatic
moiety.
Comparing epibatidine [9], (A,(+) = 0.053 nM, A,(-) = 0.073 nM) and compound 1,
(Ki = 17.4 nM), the affinity decreases dramatically. Thus the insertion of an oxygen
atom increases interatomic distances between the quaternary and pyridinyl nitrogens.
The N-N distance in (-)-9 is 5.51 A° and in 1 is 6.5 A°. These are considered two
important pharmacophores for the binding to the receptor (43) and the structural
distance between them may play an important role. In fact, by shifting the pyridinyl
nitrogen from the meta ((±)-l) to the ortho position ((±)-5), the N-N distance changes
from 6.5 A° ((±)-l) to 5.2 A° ((±)-5) and the affinity decreased 35 fold (Ki = 620.5
nM). Moreover when the aza-heptabicyclo ring ((±)-l) is substituted with an aza-
optabicyclo ring (2), the N-N distance increases from 6.5 A° ((±)-l) to 7.2 A° (2) and
the affinity decreased 50-fold (Ki = 955 nM).
The investigation of the type of substituent in the aza-octabicyclo-derivatives revealed
that the insertion of an unsaturated bond increases significantly the binding to the
receptor. In fact, compound (±)-6 showed the highest affinity (Ki ~ 2nM) within the
epibatidine derivative series. This may be due to two facts: first the presence of a
double bond in the structure limits the conformational flexibility making the
tridimensional structure of the compound similar to epibatidine (44). Compound (±)-6
contains only one rotatable bond connecting the cationic and hydrogen bond acceptor
pharmacophoric parts. This would lead to a low-energy conformer with less space
flexibility which should favour interactions with the receptor site. Second the removal
52
of any electrophilic substituent like OH (3) and F (4) from C3 position, restores the
hydrophobic core, increasing the receptor affinity. This is, in fact, what happens with
compound (±)-6.
The dramatic decrease in affinity of compound 3 (Ki > 20000 nM) compared to
compound 4 (Ki = 38.8 nM) may be due to the hydrophilicity and polarity of the
central area (which should be hydrophobic) and to hydrogen bond interactions. The
OH group's position in may considerably influence the interactions of the ligand's
pharmacophores with the receptor binding site.
(±)-6, among the three racemates investigated, is the only one exhibiting suitable
affinity towards a4ß2 nAChR subtype to be investigated by electrophysiology (see
Section below) and be used as a potential in vivo PET tracer. (±)-6 has not been
enantiomerically separated due to the high structural analogy between the enantiomers
(Figure 2). This was confirmed by molecular modeling analysis (Bisson ct al 2003
submitted): because of this analogy there should not be too high differences in the
binding affinity to the receptor.
Figure 2. Superimposition. Superimposition of the two enantiomers of
compound 6 (created with SYBYL 6.8 provided by Tripos Inc., St. Louis,
USA). Nitrogens are colored in blue, carbons are colored in white and
hydrogens are colored in cyano.
53
Toxicity studies
The mice were treated with an intravenous dose of 10 ug/mouse (20-30 g) and with the
exception of one drug-independent mouse death, all animals survived. The median
lethal dose (LD50) of compound (±)-6 (MW: 200.29) after a single intravenous
injection in mice of both sexes, observed over a period of 14 days, was thus
established to be greater than 0.5 mg /kg body weight.
Compound (±)-6 shows much lower toxicity compared to epibatidine [9] (> 50 times
less) and the innovation of the toxicology studies performed is interesting. The
intravenous dose of 10 ug/mouse was decided in advance calculating first the minimal
dose needed for a human PET investigation and then, by multiplicating this 104 times,
an initial dose with a wide range of safety should be obtained. This methodology
avoids the useless sacrifice of many mice and gives toxicology results which are
reliable and with a satisfactory safety profile.
Electrophysiology
Ligand-gated channels behave as allosteric proteins with multiple conformations and
the receptor can change state in the absence of the ligand. The "multiple allosteric
states" model describes four states, B, A, I and D (Figure 3).
B^F ^A
Figure 3. The "four-state" allosteric model. Active state (A),Basal state (B), Intermediate state (I) and desesitised state (D).
54
In the absence of an agonist the receptor is in the resting B (closed) state and a fraction
of the receptor can be in the desensitised D (closed) state according to the equilibrium
constants. When the agonist binds, the active A (open) state is stabilised first, then the
intermediate 1 (closed) state and finally with a slower time constant, the desensitised D
(closed) state. The transition from one state to another can happen in the absence of
ligand and the compounds influencing the transition constants can behave as receptor
potentiators or inhibitors (45). When an antagonist binds, the resting B (closed) state is
stabilized. On the other hand when a partial agonist binds the active A (open) state is
stabilised but will less efficacy. Thus a fraction of the bound receptor is quickly
desensitised with the channel closure. At the same concentration, then, a partial agonist
exhibits less biological response compared to a full agonist.
Electrophysiology studies compared to binding studies are based on an different
principle. The inhibition constant calculated with the competition assay (see previous
section) measure the ligand affinity toward the desensitised (closed) receptor state.
This is due to the fact that, during time incubation (1-2 h according to the radioligand
used), the receptor, initially in the active (open) state, is desensitised. With voltage
clump electrophysiology, instead, thanks to the quick measure of the current passing
through the ion channel, is possible to investigate all existing receptor states to which a
ligand may bind.
The concentrations of the ligands are indicated on the graphs (see Figures 4-8). The
compounds are dissolved in the physiological perfusion medium.
Towards the ot4ß2 receptor subtype 3-bromocytisine 8 and 3-(4'-fluorophenyl)cytisine
7 are poor agonists compared to the ACh control response at 50 uM concentration; at
lOOuM the former is a better agonist able to evoke some current while the latter
evokes hardly any current even at 100 uM. They both show no antagonistic activity
(Figure 4). Cytisine evoked partial agonist activity at 300 uM decreasing more than ten
times the 50 uM ACh control response.
At the ci3p4 receptor subtype, if we compare the ACh control response at 50 uM, 8 is
almost a full agonist at very low concentration (100 nM) whereas about 100 uM of 7 is
required to evoke the same current. Very poor antagonism activity is observed with 7
and 10 at 100 nM concentration (Figure 5).
55
16,6
Cytisine
100 nM
nAec
-32.4
-69.2
1 -105.9
10,0 15.0 0.0
100 nM
100 nM
5,0 10.0 15.0
17,18nA SOjiMACh
3.9
-88.7
-181 3"
-273.9
control
+ 100 nM BromoC
or FluoropheC
SO 10.0 15 0
20.21nA 50l,MACh
4.4
-100.6
-205.5 ~
-Î10.5
+ lOO nM Cytisine
control
0.0 50 10.O 15.0
Figure 4. 0f4p2 subtype. Agonist and antagonist activity of cytsine, bromocytisine
(BromoC) and phenylfluorocytisine (FluoropheC) towards cx$2 subtype.
lOOnMBC
0,0 50 10.0 15.0 200
iètondi I7-OI-02.1U
29 3UJ35
lOOnMFpC
100n M Cytisine
0.0 5.0 10,0 15,0 »0100 150
.333MI50|iMACh
634.7
6965.0
14564.6 // +100 nM Cytisine
U*/ control
0,0 10.0 15.0
3Î.3M1
M 50 |iM ACh
634.7
-221643
SOuMACh
-221643
00 50 10.0 15,0
Figure 5. <X3ß4 subtype. Agonist and antagonist activity of cytsine, bromocytisine
(BC) and phenylfluorocytisine (FpC) towards a3ß4 subtype.
nAnA
43.4S;47 43,45:47
4.6
\ / 1 OOn M BC
«8
\ /1042
I / -104-2
1 / SO |iM ACh
OC> 5.0 lO.O ISO
**concfc 1 7-Ol -02.t>a
lOOn M Cytisine
-
lOOn IVI FpC
•
56
50nMACh
V"''
^ control
+ 100nM BC
i 1 1
5.0 10.0 15 0
Figure 6. On subtype. Agonist and antagonist activity ofcytsine, bromocytisine
(BC) and phenylfluorocytisine (FpC) towards a7 subtype.
Towards the a-] receptor subtype 8 is an agonist whereas 7 and cytisine (10) do almost
nothing at 100 nM. At the same concentration no antagonistic activity is seen (Figure
6).
Towards the a3ß4 and a7 receptor subtypes ligands (±)-l and (±)-5 exhibit no activity at
1 uM concentration (10 pJvI and 1 mM ACh control affinity) and no high binding,
because the intensity of the ACh response did not change upon addition of the
compound.
At the a4ß2 receptor subtype at 1 \iM (10 uM ACh control) ligand (±)-l shows no
activity and no high affinity binding and ligand (±)-5 shows low partial agonism as
shown by the decrease ofACh response after addition of the compound.
With ligand (±)-6 more electrophysiology studies were performed because of its
favorable properties (high affinity and low toxicity).
Towards the a7 receptor subtype ligand (±)-6 evoked no detectable response at 1 uM
concentration (1 mM ACh control) which is confirmed by the test pulse experiment (1
uM for each ACh pulse and 10 nM ligand pulses.
At the a3ß4 receptor subtype ligand (±)-6 showed some slight activity (even without
high affinity binding) at 1 uM concentration (10 uM ACh control) which was
confirmed by the test pulse experiment (1 |xM for each ACh pulse). The current versus
time after 100 nM ligand pulses increased compared to the control values revealing
some agonistic activity. This effect was not seen with 10 nM ligand pulses. This means
that some agonism activity toward ci3ß4 receptor subtype is detected at high (±)-6
concentrations and with a concentration ratio between the control ACh and (±)-6 of
minimum 10:1.
5255
2UX
-109J
-219.4
,* 50|iMACh
DA
-329.0
control or
+ 100 nM FPC
52:55
2s«
-109J
-219.4
^ SOjiMACh
1/contr"
i 1 AH n
0.O 5.0 10.O 15.0-3294
control
+100 nM Cytisine
5255
2s-05
-1M.7
-219.4
.4. ^_ J -mo
Ofl 5.0 10.0 15.0 an
57
Towards the 0^2 receptor subtype ligand (±)-6 exhibited partial agonistic activity as
shown by the decrease of ACh response after addition of the compound at 1 uM
concentration (10 \iM ACh control). The test pulse experiment performed with 10 and
100 nM ligand pulses (1 uM for each ACh pulse) showed some antagonism activity as
the current versus time increased compared to the control values. This effect is
concentration dependent. In both experiments with 013ß4 and 014^2 receptor subtypes the
displacement of (±)-6 by ACh was slow: both receptors returned slowly to the normal
response under control conditions (Figure 7-8).
35 10(iM ACh
/
-1624 1 /-3209 1
/ 1 1
35 1mM lOvtMACh
-39 1
n r~
1624 1
l
32« 1
v
4794 1 i I I
35
-30 1
10 11M ACh
\62& 1
3209 1
.
1 1 1
1Û0 150
ttcandi 22-*-02bj
100 150
'xtïôrïdi 22 8-02bj
Figure 7. 04ß2 subtype. Activity of 1 |xM of 6 towards gc^2 subtype after
the application of 10 p.M ACh control.
78,« OR2 control
nA
766
OR2 control (waih)
If— r~-~
rif r- m— f- 1— r*--
r"~l
I)
1 I*
v1
4
1 uM Ac h trst pulse* lui<A Ach + 3 3b IO nM test pulse luM Ach te si pul &e
00 150
seconds
22-S-02 ba
Figure 8. 0^2 subtype. Activity Pulse test experiment with 10 nM of 6 (33b)
and 1 p,M ofACh towards a$2 subtype after the application of 1 p,M ACh control.
This may depend on the different receptor-ligand dissociation constant of (±)-6 and
ACh or on the fact that a fraction ofthe receptor with the time is not more in the active
state (open) but in the desensitised state (closed). Compound (±)-6 behaves as a full
58
agonist and a partial agonist respectively towards <X3ß4 and a4ß2 nAChR subtypes. This
means that in the case of the neuronal subtype, after (±)-6 binding, the receptor in its
active (open) state is quickly desensitised with higher rate constant than with the
ganglionic subtype. Thus this would explain the results obtained with the 10 nM test
pulse experiment. In fact, in the case of the ganglionic receptor the time recovery of
ACh response is faster.
The insertion of a bromine atom at the 3-position of cytisine (bromocytisine, 8)
enhances the biological response to the a^ subtype. The full agonism of 8 stabilizes
the receptor A (open) state increasing the risk of getting toxic side effects to the
interaction with the ganglionic nicotinic subtype. Some agonistic activity of ligand 8
was observed on a4ß2 and a7 receptor subtypes which is concomitant with the low
nanomolar affinity obtained by binding studies (Table 1). The insertion at the 3-
position of a p-fluorophenyl group (7) reduces dramatically the response of all receptor
subtypes. This is in accord with the lower affinity towards a4ß2 and a7 neuronal
receptors (Table 1) due to the possible energetically unstable orientation of the p-
fluorophenyl group in the receptor.
The epibatidine analogues (±)-l and (±)-5 showed no considerable biological activity
towards all receptor subtypes, which may be the consequence of the low affinity of
both compounds (Table 1).
Ligand (±)-6, instead, was more carefully studied because of its nanomolar affinity
(Table 1). Compared to the partial agonism towards the a4ß2 receptor subtype, no
appreciable biological response was found towards a.3ß4 and a7 receptor subtypes.
Thus compound (±)-6 is selective for neuronal subtypes stabilizing, in a concentration-
dependent manner, the open active as well as a the closed a4ß2 receptor state.
The four-state allosteric model may also explain the difference between the partial
agonism of (±)-6 and the full agonism of dechloroepibatidine (DClEpi) (28) towards
a4ß2 nAChR subtype. Spang et al. demonstrated that DClEpi agonism is due to the
stabilization of the active A (open) state even better than ACh. As a consequence, a
loss of agonism reflects a reduction in the stabilization of A state. The partial agonism
of (±)-6 exhibiting a lower biological response at the same concentration can be only
explained by the fact that the ligand is stabilizing the A state in a less extent than
DClEpi. This would then lead to a faster receptor desensitisation with the consequence
59
of channel closure and antagonism characteristics like the non depolarization of the
oocyte membrane.
The full and partial agonism towards a certain subtype is strictly dependent on the
orientation of the ligand in the binding site region. As a consequence, this orientation
affects, by allosteric movements, the opening and closure of the ion channel. Starting
from DClEpi structure, for example, the enlargement of the aliphatic cycle size and the
insertion of a double bond to get (±)-6, lead to different ligand interaction and
orientation in the binding site region. Thus, the way the ligand binds to the receptor,
influences the equilibrium constants between the different receptor conformational
state. This means that, in the case of DClEpi and (±)-6, respectively the active and the
desensitised state are more stabilized, showing full and partial agonism towards a4ß2
nAChR subtype.
In conclusion, the sum of informations, coming from in vitro binding and
electrophysiology studies, are essential to understand the relationship between ligand
structure and biological activity. The increase or decrease of ACh control signal
intensity, sometimes found after ligand application, may be due to the fact that the
conformational states of the receptor are changing during time, with fractions of the
receptor existing in different states. Hence, this would confirm that nAChRs are
membrane proteins with significant allosteric connections between the binding site and
the ion channel receptor regions.
Experimental Procedures
Membrane Preparation
Frozen male Sprague-Dawley rat brains (20g, RCC Ltd, Füllinsdorf, Switzerland and
Charles River Labs, Sulzfeld, Germany) were homogenized in 10 volumes of ice-cold
(4°C) sucrose buffer (0.32 M sucrose, lOmM Tris/acetate-buffer pH 7.4, 0.02% NaN3)
with a polytron (PT-1200 C, Kinematica AG, Littau, Switzerland) for 1 min at setting
4.The homogenate was centrifiiged at 1000g for 15 min (4°C) and the pellet was
resuspended in 5 volumes of sucrose buffer, homogenized and centrifiiged again at
1000g for 15 min (4°C). The resulting supernatants were combined and centrifiiged at
6667g for 20 min (4°C). The pellet was resuspended in ice-cold incubation buffer
(5mM Tris/acetate-buffer pH 7.4), homogenized and centrifiiged at 6667g for 20 min
(4°C). The pellet was resuspended in incubation buffer and stored at -70°C. On the
60
day of the assay, the membranes were thawed and the protein concentration
determined by Bio-Rad Microassay with bovine serum albumin as a standard
(Bradford 1976).
Competition Binding Assays
Binding assays of (±)-l, 2, 3, 4, (±)-5, (±)-6, 7, 8 for the a4ß2 and ct7 neuronal nicotinic
receptor subtypes were performed at the final volume of 0.2 ml containing 100 ug of
protein (from membrane preparation of frozen Sprague-Dawley rat brains) in a BSS
buffer at pH 7.4. The concentration of [3H]-cytisine (72000-59600dpm/pmol) was
2nM. After lh at 0°C, the samples were vacuum-filtered on glass fibre filters
(Whatman GF/C) which were then washed with ice-cold buffer (2*4ml). Non-specific
binding was estimated in the presence of 0.1mM nicotine. Packard Ultima Gold was
the cocktail used for liquid scintillation counting with a Tri-Carb 2200CA analyser.
The concentration of [125I]-alpha-bungarotoxin (213839-102091 dpm/pmol) was
0.5nM. After 2h at 37°C, the samples were vacuum-filtered on glass fibre filters
(Whatman GF/B), which were then washed with ice-cold buffer (3><4ml). Non-specific
binding was estimated in the presence of O.OlmM alpha-bungarotoxin. The counter
analyser was a Cobrafl autogamma from Packard. The binding results from the counter
were analysed using the Kell Programme from Biosoft using respectively Kd = InM
for (-)-[3H] Cytisine (a4ß2 subtype) and Kd = 0.7 nM for [125I]-a-Bungarotoxin (cc7
subtype).
Toxicology
The toxicology studies were performed by RCC Ltd Toxicology Division (Fiillinsdorf
/ Switzerland).
Electrophysiology
All the cDNA constructions were cloned from rat. For the a7, a3ß4 and cl$2 nAChRs
reconstitution in Xenopus oocytes vector pcDNAIneo with RSV promoter was used.
Oocytes were harvested from female Xenopus leavis, prepared and injected as
previously described [42,43]. Oocytes were injected with rat cDNA combinations cl$2,
oi3ß4 and a7 and the physiological properties of the different subtypes were examined
2-3 days following the injection using a dual-electrode voltage clamp.
Electrophysiological recordings were performed using a dual-electrode voltage clamp
61
apparatus (Geneclamp, Axon Instruments, Forster City, CA) [70]. During the
experiments, oocytes were continuously flooded with control solution containing 82.5
mM NaCl, 2.5 mM KCl, 5 mM HEPES, 2.5 mM CaC12, 1 mM MgC12, pH 7.4
(NaOH). Perfusion was gravity driven at roughly 6 ml/min and drugs were applied by
electromagnetic valves (general valve type III) controlled by computer. Unless
indicated, cells were held throughout the experiments at -100 mV. Data are
represented with their respective standard error ofmean (SEM).
Significance
In recent years, it has become clear that the neuronal nicotinic acetylcholine receptors
(nAChRs), ligand gated ionic channels, may play a role in a variety of diseases,
including Alzheimer's disease (AD), anxiety disorders and nicotine addiction. The
alkaloid epibatidine (EPB), originally isolated from the skin of the Ecuadorian poison
frog, Epipedobates tricolor, by Daly and co-workers, is a very specific agonist of the
neuronal nAChR. However, the toxicity of this molecule prevents its therapeutic and
even diagnostic use in clinical medicine. To develop new ligands selective for distinct
nAChR subtypes with a satisfactory safety profile, new epibatidine and cytisine
analogues have been synthesized and the in vitro binding affinity towards the 0^2 and
a7 neuronal nAChR subtypes was investigated. Voltage clamp electrophysiology was
performed with these compounds on the neuronal cl$2 and a7 and on the ganglionic
ci3ß4 nAChRs reconstituted in Xenopus oocytes.
Compound ,(±)-6, with the right key structural features for binding: the positively
charged nitrogen atom for ionic or hydrogen bond interactions, the lone electron pair
of the pyridine nitrogen as a hydrogen bond acceptor and the hydrophobic area of the
aliphatic octa-cycle, demonstrated high affinity (2 nM) and partial agonism towards
a4ß2, subtype selectivity and low toxicity in mice (LD50 > 0.5 mg/kg body weight).
This compound shows good characteristics to be developed as a positron emission
tomography (PET) tracer for imaging nAChRs, labeled with HC or 18F.
62
Acknowledgments
This work was supported by the Swiss National Science Foundation, nr. 3100-53'638
toDB.
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67
CHAPTER 4
Homology Modeling, Dynamics of Rat and Human Extracellular
Domain of a4p2 and al Neuronal Nicotinic Acetylcholine
Receptor Subtypes
submitted to Journal ofComputer-aided Molecular Design
William H. Bisson^ Gerrit Westera,*'*
P.Augustus Schubiger,^ and Leonardo
Scapozza,s
T Centerfor Radiopharmaceutical Sciences, Swiss Institute of Technology Zurich, Paul
Scherrer Institute, Villigen and University Hospital of Zürich, Rämistrasse 100, CH-
8091 Zürich, Switzerland, and § Pharmaceutical Biochemistry, CHAB, Swiss Institute
ofTechnology Zurich, Switzerland
ABSTRACT
In recent years, it has become clear that the neuronal nicotinic acetylcholine receptor
(nAChR) is a valid target for a variety of diseases, including Alzheimer disease,
anxiety and nicotine addiction. As for most membrane proteins, information on the
three dimensional structure of the nAChR is limited to data from electron microscopy
with a resolution that makes difficult the application of structure-based design
approaches to develop specific ligands. Recently, the first high-resolution structure of a
soluble acetylcholine binding protein (AChBP), extracted from snail neurons, and
homologous to the ligand extracellular binding domain of nAChR, has been
determined. Based on this high-resolution crystal structure, the homology-models of
the extracellular domain of the neuronal rat and human a4ß2 and a7 nAChR subtypes
(the most present subtypes in the brain) were built and their stability assessed for the
first time with Molecular Dynamics (MD). The resulting models may be used for
Molecular Docking to provide a valuable framework for structure-based design of
specific a4ß2 nAChR subtype ligands, which should improve therapeutic and
diagnostic applications for diseases involving this receptor.
* Author for correspondence:Tel: xx41 1 255-35-60, Fax: xx41 1 255-44-28, email:[email protected]
68
Introduction
Ion channels are membrane proteins with an important physiological and
pharmacological role. The nicotinic acetylcholine receptor (nAChR) is a ligand-gated
ion channel located mostly in the central and peripheral nervous system and
neuromuscular junction. In particular, neuronal nAChRs are present in presynaptic
regions to mediate acetylcholine neurotransmission (1) and in the postsynaptic
membrane to propagate via acetycholine the nerve pulse through the neurons.
Moreover, it is composed of five polypeptide chains (subunits) which form a cation
permeable pore and build up homopentamers (identical subunits) or heteropentamers
(different subunits). The binding sites are located at the interface between two
extracellular subunits and their number differ from subtype to subtype. Thus, different
nAChR subtypes exist according to the type of a and ß composition and the open of
the channel can be caused by other agonist than acetylcholine like nicotine,
epibatidine (2) or by synthetic compounds (3,4). Reconstitution experiments
performed in host systems such as Xenopus laevis oocytes and/or transfection in cell
lines together with the combination of site-directed mutagenesis and single-channel
studies have been performed. The results obtained have demonstrated that the
pentameric assemblies (a4)2(ß2)3 and (a7)s are the most present subtypes in the brain
(5). In addition, the identification of many neuronal nAChR genes has given new
imputs to understand the mechanism of neurotransmission which correlates
neurological diseases and nAChRs (6). In the last years, neuronal nAChRs are become
an important drug target for a variety of diseases including Alzheimer (7,8).
Considerable effort has been made to understand and investigate the structure of
nAChRs especially at the binding site region. Due to crystallization problems, as for
most membrane linked-proteins, the only available tridimensional structure
information of nAChRs is the low-resolution data from electron microscopy (9).
Unfortunately, this is not enough to investigate the conformational changes due to
ACh binding causing the opening of the ion channel.
An high-resolution structure of the acetylcholine-binding protein (ACh-BP) (10),
isolated from snails has been recently reported and showed an homopentameric
structure. This protein is produced and stored in glia cells and released into the
synaptic cleft in an acetylcholine-dependent manner to modulate neurotransmission
69
(11). AChBP binds nAChR agonist such as acetylcholine, nicotine and the competitive
antagonist a-bungarotoxin (11) and reveals a significant sequence homology with the
extracellular part of the nAChR subunits (26% identity with al subtype)(12).
Moreover, AChBP exhibits pharmacological properties and ligand affinity (es.: a-
bungarotoxin) similar to the a7 homopentamer suggesting three-dimensional structural
similarities (13,14). Therefore, AChBP can be used as template for the 3D-structure of
the N-terminal domain ofa and ß nAChR subunits.
Many protein sequences are now available in specialized databases and homology
modeling is an useful tool to construct models starting from a known target sequence
and a known 3D-protein template. The sequence identity between target and template
should be more than 22 % to be able to get a reliable homology sequence alignments
(15).
In the present work the homology-models of the extracellular domain of the neuronal
rat and human (a4)2(ß2)3 and (al)$ nAChR subtypes were built and their stability
were assessed with Molecular Dynamics.
Methods
Sequence Alignment and Homology Modeling. The tertiary structure prediction of
the amino-terminal extracellular domain of human al, rat a7, human a4 and human ß2
nAChR subunits was performed with the program SwissModel (16-18) using the First
Approach Mode with user-defined 3D-templates including energy minimization
performed with the program Gromo 96.
For the amino-terminal extracellular domain of human al, rat al and human a4,
human ß2 subunits, the 3D-templates with their sequences used were the AChBP
monomer (10), the amino-terminal extracellular domain of chick al, rat a4 and rat ß2
subunits (12) respectively. The 3D-templates rat a4ß2 and chick al pentamers were
retrieved from the Ligand-Gated Ion Channel database of the Pasteur Institute, France
(12).
The three-dimensional models of the pentamer of neuronal rat and human (a4)2(ß2)3,
rat (a7)5 and human (a7)5 nAChR subtypes were assembled using SYBYL 6.8 (Tripos
Inc. (St. Louis, USA)).
70
The prep file of acetylcholine and its insertion into the AChBP dimer binding cavity
was performed using the program ANTECHAMBER and LEAP of AMBER7
(Assisted Model Building with Energy Refinement, University of California (USA)).
All the assessment of the geometry of the model such as protein bonds, bond angles
and torsions etc. were performed using the program PROCHECK (19). The three-
dimensional models of the dimer and pentamers were visualized and manual refined to
correct non allowed geometrical parameters with the software SYBYL 6.8 (Tripos
Inc., St. Louis, USA).
Energy Minimization and Molecular Dynamics. The water boxing, charge
neutralization with Na+ counterions, energy minimization, molecular dynamics and the
correspondent analysis e.g. RMSD (root mean square deviation) graphics versus time
(picosecond) and versus residue number of the models, were performed using program
AMBER6.
The Amber force field (20) all atom parameters were used for the protein and the Na+
ions. The minimization protocol consisted in 1000 cycles of steepest descent followed
by conjugate gradient method until the root-mean square deviation (rmsd) of the
Cartesian elements ofthe gradient reached a value smaller than 0.15 Â.
Periodic boundary conditions were applied. The dynamic protocol is divided in three
steps MDl, MD2 and MD3. During all dynamics steps the reference temperature at
which the system had to be kept was 300 °K according to Berendsen's coupling
algorithms (21). Although, the initial temperature for MDl, MD2 and MD3 was set
respectively at 0, 150,300 °K. The time step for all three dynamics procedures was
0.002 picosec (ps). For minimization and molecular dynamics, the primary cutoff
distance for non bonded interaction was set at 9 Â.
Regarding the molecular dynamics protocol used, the first (MDl) aimed the
equilibration of water molecules and ions of the water boxed and charge neutralized
model. An initial velocity was given to the system and then trajectories were allowed
to evolve in time according to Newtonian laws keeping the model protein fixed. The
number of dynamics steps was 7500 corresponding to 15 ps.
15 ps of constant volume dynamic (MD2) was performed on the all system to adjust
density to a value of 1. In the third step a 500 ps constant pressure dynamic (MD3) at 1
atm was applied without any constraint to finally assess conformational stability.
71
All geometry quality assessment of the models, at different time points, were made
using the program PROCHECK (19).
Results and Discussion
Sequence Alignment, Homology Modeling.
The sequence alignment, showed in Figure 1, indicates the homology and the identities
between AChBP and the amino-terminal extracellular domain of the human al, the
amino-terminal extracellular domain of chick a7 and rat al and the amino-tcrminal
extracellular domains of rat <x4, rat ß2 and human a4, human ß2. The correspondent
values in percentage are shown in Table 1. This reveals a high homology between
same nicotinic subtypes of different species which confirms a common origin point
during ligand gated ionic channel superfamily evolutionary tree (22).
JSl_
tl_ti_
ail
Figure 1. Sequence alignment. Multiple sequence alignment homology performed, a
= AChBP; al = o4r; a2 = o4h; bl= ß2r; b2 = ß2h; cl= a7c; c2 = a7r; c3 = a7h. r = rat;
h = human; c = chick.
72
TEMPLATE
AChBP a 7c a\x in*
28.8 93.7 44.9 40
28.3 91.2 45.4 39.9
24.9 45.9 97.6 53.4
24.6 40.4 54,3 98.5
c, chick: r, rat: h, human.
Tablel. Percentage of sequence identities computed with the LALIGN
program (41 )
The AChBP homopentamer has five binding sites. Each binding site is formed in a
cleft made of different loops, part of the principal subunit face (plus side), and a series
of ß-strands, part of the complementary adjacent subunit face (minus side). The
residues involved are in agreement with the ones identified in the nAChR binding site
by photoaffinity labeling and mutagenesis studies (23-35). The plus/minus side
interface consists on the plus side formed by amino acids Tyr 89 (loop A), Trpl43
(loop B), Tyr 185, double cysteine 187,188 and Tyr 192 (loop C) and on the minus
side including aminoacids Trp 53, Gin 55 (ß-strand D), Arg 104, Val 106, Leu 112,
Met 1 14 (ß-strand E), Tyr 164 (loop F) forming a cavity with a top (Tyr 89, Tyr 185,
Tyr 164, Trp 53), a bottom (Arg 104, Val 106, Leu 112) and walls (Tyr 192, Trp 143,
the main chain closed to residue 145, the side chains of Met 114, Gin 55, double
cysteine 187,188). The homology of the four nicotinic models with AChBP, at the
residual level, involves mostly the plus side than the minus side (Figure 1). The
aromatic residues Tyr 89, 192 and Trp 143 of the plus side and Trp 53 of the minus side
are conserved in all a and ß subunits of different species. This would confirm the
importance of cation-7i interactions between the ligand and the protein receptor. The
double cytisine 187.188 present in the plus side of AChBP differentiates a from ß
subunits. Regarding the AChBP minus side the residues Arg 104, Val 106, Leu 112,
Met 114 arc poorly conserved within the different nicotinic subunits. In the binding
site region of rat (a4)2(ß2)3 model the residues expected to be involved in the ligand
binding on the a subunit and ß subunit are respectively Tyr 91 (loop A), Trp 149 (loop
B), Tyr 188, two cysteine 190,191, Tyr 195 (loop C), Trp 55. Thr 57 (ß-strand D), Val
73
109, Ser 111, Phel 17, Leu 119 (ß-strand E). Similar results are also obtained with the
other models. Thus, the residues on the a subunit (plus side) are conserved within
different subunits of the same species and within the same subunit of different species.
As a consequence, this would confirm what has been found by Le Novère et al. with
the chick (al)5 model (12). Thus, the plus side in the interface of adjacent subunits is
the principal component involved in ligand binding. On the other hand, the minus side
behaves as the complementary component and may be the side responsible for subtype
specificity.
Figure 2 shows the comparison of the binding sites of AChBP, rat (a7)5 and (a4)2(ß2)?
nAChR subtype at the interface between the two adjacent subunits. The homology
with AChBP at the primary structure level, as mentioned before, involves mostly the
aromatic residues (Trp 143, Tyr 89, 185 and 192) and the doublc-cysteine 187,188 of
the al and a4 plus side. Whereas there is low homology between AChBP and the
residues of the al and ß2 minus side with the exception of Trp 258 (Trp 53 of the
minus side) which is conserved in the nicotinic models too. The AChBP residues of
the plus side Thr 144 and His 145 (Figure 2) are replaced, respectively, in the al
subunits and in all nicotinic subunits (Figure 1), with amino acids like Ser and Tyr,
which keep similar functionality. At the 3-D level, within all models, the binding site
is at the interface between two adjacent subunits with a plus side formed by different
loops of one subunit and a minus side formed by a series of ß-strands of the other
subunit. This confirm what was discovered in the past from the modeling of putative
three-dimensional structure of the nAChRs (36,37). The conserved aromatic residues
(Trp 143, Tyr 89, 185 and 192 of the plus side and Trp 53 of the minus side of
AChBP), showed in Figure 2, part of different loops, form an idrophobic pocket in the
inner part of the cavity, which is amenable for cation~7r interactions. On the other hand,
the poorly conserved residues of the AChBP minus side (Gin 260, Leu 317 and Met
319), visible in Figure 2, part of different ß-sheets. cover the aromatic pocket and arc
more involved in hydrogen bonds with the ligand. Thus, the low homology found may
explain the subtype specificity role of the complementary minus side. The ACh
binding site is located at the interface between two subunits and the ligand must
penetrate into a gorge to interact specifically with the receptor and give the biological
response. The doublc-cysteine loop seems to be located at the entrance of the cavity
regulating and preventing the access, as in
74
Figure 2. Superimposition. Superimposition of the binding site of AChBP (atom
type color) and rat (a4ß2) pentamer model (orange) and rat (a7) pentamer model
(green) The residues are labeled in white (AChBP), yellow (a4ß2) and cyano (a7).
In the case of residue conservation within the three models only the AChBP
aminoacid side chain is shown.
AChBP, of the ligand according to steric hindrance and of the solvent from outside,
respectively.
Molecular Dynamics.
The insertion of acetylcholine into the binding cavity of the AChBP dimer took into
account of the tridimensional orientation of N-2-hydroxyethylpiperazine-N'~2-ethane-
sulphonate (HEPES) with its cation-Tr. interactions. Each binding sites of the AChBP
pentamer contains a molecule of HEPES coming from the crystallization procedure.
This buffer molecule has a positively charged nitrogen similar to known nicotinic
75
ligands which stacks onto Trp 143, making cation-7i interactions which is also a very
important interaction for the binding of ligands to nicotinic receptor (38,39). Then the
AChBP dimer with and without acetylcholine was submitted to the described
minimization and MD protocol. Figure 3 indicated the RMSD as a function of time
and clearly shows that a plateau, representing binding orientation stable over time, was
reached already after 50 ps. The comparison of the structures at different time of the
MD with the starting structure revealed no major changes in the overall conformation.
The low RMSD (1.5 Â) indicates that the starting structure represents a stable
conformation and the MD protocol allows us to assess the stability of the models.
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0
0 50 100 150 200
time (ps)
Figure 3. Acetylcholine. RDMS graphic versus time (213.5 ps) of
acetylcholine of the (AChBP + ACh) dimer mode.
The models rat and human (a4)2(ß2)3, rat (a7)5 and human (a7)2 were submitted to the
same MD protocol to assess their stability. The RMSD graphics versus time are shown
In Figure 4.
The RMSD profile clearly shows that all molecules reached a plateau and thus
conformational stability. As expected, the RMSD values of the nicotinic models were
found to be higher between 2.5-3 Â then the values of the crystal structure AChBP
which were around 1.5 Â. The RMSD graphics versus residue number, after dynamic
were made with all the models revealing that the major movements of the protein,
during the dynamic steps, do not involve most of the aminoacids which form the
binding site cavity and participate in making important interactions with the ligand.
The AChBP dimer complexed with acetylcholine was analyzed. The importance of the
76
plus and minus site forming the cavity and surrounding the acetylcholine molecule was
confirmed.
»H,
4 ^ ^
] ^
1ç
-V
> «
35
2,5
v> 2 !E
- ^ /**v^/^w*-v*'Wr
15 f* ."V**.i ***# -»^ •*'»-i» ^*^"4
t
05
100 200 300 400
time (ps)
B
500
Figure 4. Models. A: a4ß2 rat pentamer model, B a4ß2 human pentamer model,
C' RDMS graphics versus time (picosecond) Dark blue = AChBP pentamer, orange
= AChBP dimer, violet = human (a4ß2)s red - human (<x7)2, green= rat (<x7)<;,
light blue = rat (a4ß2)5 .All atoms of the models were submitted to MD The
observed increase of RDMS after 400 ps with the ha7 dimer can be expecteddue to the fragility of a dimer model submitted to long dynamics
*<* ""T
Figure 5. AChBP with ACh model. Binding site region of the AChBP
dimer modeled with ACh. In orange the residues of the (+)-side and in
green the residues of the (-)-side. Dashed lines represent H-bonds.
Figure 5 shows the presence of cation-7t interactions between the positively charged
quaternary ammonium group of ACh and Trp 143, Tyr 89,185,192 of one side and the
hydrogen bond between the carbonyl oxygen of ACh and the N-H of Met 114 (2.678
Â) of the opposite complementary side. Those type of non-covalent interactions were
proved with site-directed mutagenesis to be important also in the binding of ligands to
nicotinic receptors (22,40). During MD, cation-7r interactions remain stable and this is
shown from the low RMSD deviation of acetylcholine during the MD (Figure 3) and
the analysis of the structure at different time.
In the case of the AChBP dimer without acetylcholine, Trp 143 (loop B) and Tyr 192
(loop C) shift toward the inner of the cavity during MD (Figure 6) On the other hand,
in the AChBP dimer with acetylcholine, Trp 143 and Tyr 192 do not show a significant
78
shift. Hence, this proves that the observed shift is due to the dynamic, although, the
movement of Trp 143 and Tyr 192 toward the inside of the binding cavity, is strictly
dependent on the emptiness of the cavity itself. In fact, looking at the empty binding
site region of rat and human (a4)2(ß2)3,
rat (al)$ and human (a7)2 models after
dynamics, we confirmed the same shift direction of the residues Trp and Tyr, which
are conserved residues within the different nicotinic a-subunits.
Figure 6. Trp 143 and Tyr 192. Tridimensional shift of Tyr 192 and Trp
143 from X-ray with HEPES inside (red) and after dynamics in the AChBP
dimer without ACh model (green) and in the AChBP dimer with ACh
(colored by atom type) model (orange).
Conclusions
The high homology between the different nicotinic subunits from different species has
allowed us to build the models of the extracellular domain of the rat and human
(a4)2(ß2)3 and (a7)s nicotinic receptor subtypes which are the most present in the
brain. These models were for the first time successfully assessed by Molecular
Dynamic and show conformational stability over time. Hence, they may be useful,
79
with longer dynamics, to investigate the overall tridimensional movements of the
pentamer with or without the presence of the ligand in order to understand the
allosterism between the different binding sites and the kinetic of the extracellualar
domain ofthe rat and human receptor subtypes.
These models will be used for understanding ligand-binding data towards rat a4ß2
nAChR subtype aiming at improving therapeutic and diagnostic applications for
diseases involving this cationic channel receptor.
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83
CHAPTER 5
Molecular docking to investigate ligand selectivity for the
acetylcholine binding site of the rat a4ß2 and a3ß4 nicotinic subtypes
submitted to The Journal ofMedicinal Chemistry
William H. Bisson,* Gerrit Westera, ** Linjing Mu, * P.A. Schubiger,î and Leonardo
Scapozza,§
* Centerfor Radiopharmaceutical Sciences, Swiss Institute ofTechnology Zurich, Paul
Scherrer Institute, Villigen and University Hospital ofZürich, Rämistrasse 100,CH-
8091 Zürich, Switzerland, and§ Pharmaceutical Biochemistry, CHAB, Swiss Institute
ofTechnology Zurich, Switzerland
ABSTRACT
The ligand gated ionic channel nicotinic acetylcholine receptor (nAChR) is an
important target for a variety of diseases like Alzheimer and nicotine addiction. To
explore the therapeutic and diagnostic potential of small molecules binding to nAChR,
the issue of subtype selectivity has to be addressed. Recently, the first high-resolution
structure of a soluble acetylcholine binding protein (AChBP), extracted from snail
neurons, and homologous to the ligand extracellular binding domain of nAChR, has
been determined. Based on this high-resolution crystal structure, the homology-models
of the extracellular domain of the neuronal a4ß2 and ganglionic a3ß4 rat nAChR
subtypes were built. In this work, a series of nicotinic ligands were docked into the
modeled binding site cavity of both receptors. The structure-activity relationship was
investigated to understand the selectivity of the ligands for the acetylcholine binding
site of the rat a4ß2 and a3ß4 nicotinic subtypes. The docking into a4ß2 was
characterized by two distinct clusters of ligand binding orientations named clusters 1
and 2. High affinity, toxic ligands like epibatidine and dechloroepibatidine docked into
cluster 1 with the charged tertiary amino group forming a 7i-cation interaction with Trp
147 and establishing a characteristic H-bond with Lys 285. The non-toxic ligands such
as acetylcholine, (S)-A-85380 and 33bMet docked into cluster 2 with the same n-
cation interaction but with the rest of the molecule occupying a different moiety of the
binding pocket. Molecular docking into a3ß4 subtype showed that epibatidine, which
reveals in vivo peripheral side effects, is indeed a representative template for ligands
84
with affinity towards a3ß4 subtype. The ranking scores of the docked molecules
indicate the existence of structural-dependent subtype selectivity and shed light
towards the design of specific and selective a4ß2 nAChR subtype ligands with better
clinical and safety profile.
* Author for correspondence:Tel: xx41 1 255-35-60, Fax: xx41 1 255-44-28, email:[email protected]
85
Introduction
The nicotinic acetylcholine receptor (nAChR) is member of the "cys-loop"
superfamily of ligand- gated ion channels which comprises also GABAa,c, glycine and
5-HT3 receptors (1,2). In the central nervous system neuronal nAChRs are located
post- and pre-synaptically or even on axonic areas of the neuron (3). It mediates
acetylcholine neurotransmission (4) at the neuromuscular junction, autonomic ganglia
and at the central nervous system. In particular, nAChR are composed of five identical
(homopentamers) or different (heteropentamers) polypeptide chains (subunits).These
subunits, forming a cation permeable pore around an axis perpendicular to the
membrane, characterize different subtypes. The binding sites are located at the
interface between two extracellular subunits and their number differ according to the
subtype. As a consequence, in vertebrates the combinatorial assembly of al-10, ßl-4,
y, 5, e subunits generates a variety of receptors with different electrical and binding
characteristics (5). The existing different neuronal nAChR subtypes can be stimulated
by nicotine, epibatidine (6) or by synthetic compounds (7,8). Recently, it has been
demonstrated that, neuronal nAChRs, due to their important physiological and
pharmacological role, can be a useful target against a variety of diseases. These
diseases include Alzheimer, anxiety and nicotine addiction (5,9). Thus, the
development of nicotinic brain specific ligands is become, with the years, an ambitious
issue to aim for. Despite of this, subtype selectivity still remains one of the biggest
problems to overcome. Epibatidine has one of the highest binding affinity for the
neuronal a4ß2 subtype. On the other hand, it shows potent agonist activity at
sympathetic receptor like ganglionic (g.3 containing) and neuromuscular (oclPlYfi)
nAChR subtypes (10,11). The activity of epibatidine at the peripheral nAChRs is,
apparently, responsible for the in vivo observed marked hypertension and muscular
paralysis (11). Hence, the use of epibatidine results in a very limited therapeutic index
that has precluded its clinical usefulness as an analgesic. In conclusion, many toxic
clinical side effect may then be originated from the non selectivity of certain ligands
toward specific subtype receptors.
To address the selectivity issue structural informations have been seeked for long time.
The acetylcholine-binding protein (ACh-BP) crystal structure has been recently solved
(12). It is a soluble homopentamer protein produced and stored in glia cells and
86
released into the synaptic cleft to modulate synaptic transmission (13). AChBP binds
nAChR agonists and competitive antagonists such as acetylcholine, nicotine, d-
tubocurarine and a-bungarotoxin (13-14). The sequence identity between AChBP and
a, ß nAChR subunits is significative (23-26%). Therefore, AChBP can be exploited as
a template of the N-terminal domain, involving the binding site, of a and ß subunits
of nAChRs. Recently, the homology-models of the extracellular domain of the
neuronal rat (a4)2(ß2)3 and the ganglionic rat (a3)2(ß4)3 nAChR subtypes have been
published (14,15).
In the present work we used the binding site of both models to perform Molecular
Docking. A series of nicotinic ligands were docked into the binding cavities to
investigate the selectivity towards the neuronal and the ganglionic nicotinic subtypes.
Material and Methods
Molecular Modeling. The 3D-templates of rat a4ß2 and a3ß4 pentamers were
retrieved from the Ligand-Gated Ion Channel database of the Pasteur Institute, France
(14,15).
Starting from the downloaded templates, the three-dimensional models were refined
and geometrically improved with the software SYBYL 6.8 provided by Tripos Inc. (St.
Louis, USA). The obtained models were then water boxed, counterions (Na+) were
added to have a system with neutral charge and energetically minimized using program
version AMBER 6 (Assisted Model Building with Energy Refinement) provided by
University of California (USA) under periodic boundary condition. During the
different steps, the stereochemical quality of the model were assessed using the
program PROCHECK (16).
Preparation of Three-Dimensional Database of the ligands and Molecular Docking.
The nicotinic ligands to be docked were selected (Figure 1) covering a range of affinity
for the rat a4ß2 nAChR subtype from picomolar to micromolar (17,18) (Table 1). The
three-dimensional structure of the compounds was built with the program SYBYL 6.8.
Hydrogen atoms were added and Gasteiger atomic charges were calculated. Energy
minimization was performed to optimize the conformation using the Tripos force-field
implemented in SYBYL 6.8. The final coordinates of each compound were saved as a
mol2 file and all files were then stored in a single database. The docking of all
87
nicotinic ligands on both subtype models was performed using the program FlexX
(19,20) implemented in SYBYL 6.8, which used flexibly positioning for the ligands to
be placed into the active site based on the principle of shape, electrostatic and
hydrophobic/polar complementarity. A time calculation of ~ 3 min per compound was
required to dock the ligand in the flexible mode. For each ligand the ten best
energetically docking orientations were calculated. Several individual scoring function
are used to predict the affinity of ligands in candidate complexes. Screened compounds
were ranked based on consensus scoring function CScore (21). This allows a multiple
approach for the evaluation of ligand receptor interactions, that is more robust and
more accurate than any single approach (22). By means of CScore the reliability of the
suggested binding orientation was assessed.
Results and Discussion
Molecular Modeling and Docking
The neuronal rat (a4)2(ß2)3 and the ganglionic rat (a3)2(ß4)3 nAChR subtypes were
retrieved from Institute Pasteur database (14,15). The two models were refined and
geometrically improved compared to the starting templates. The number of cis-
configuration of non-proline amino acids and D-chiralities were eliminated and the
number of bad contacts were significantly reduced. Thereafter the models were water
boxed, counterions were added to get charge neutrality and energetically minimized.
The percentage of residues in the Ramachandran disallowed region, obtained by
PROCHECK (16), are 0.9% and 4.5% for the (a4)2(ß2)3 and (a3)2(ß4)3 nAChR
subtype respectively, compared to 0.6% and 5.1% ofthe beginning templates.
The inspections of the dimers (a4ß2)2 and (a3ß4)2 (Figures 2-3) at the interface
between the two adjacent subunits where the binding site is located revealed high
structural similarity with the starting template.
In both rat dimer models, in fact, the (+)- and the (-)-side are respectively represented
by the a (different loops) subunit and by the ß (a series of ß-strands) subunit which
form the binding site cleft. The homology at the residue level involves mostly the (+)-
side, while the residue forming the (-)-side are less conserved. This indicates that the
ligand may bind and have different interactions according to the subtype involved
especially at the ß subunit region.
88
CH3 N^^
A-84543o
N "OA-85380
\
CH3
°X1N
A-84543 py5
NI
CH3N
A-84543
33bMet
N+ O
Acetylcholine
O- XN»N
\CH
NI
CH3
/^Cl
A-84543 py6 Cl
A-98795
s?N
A-84543 p
Dechloroepibatidine
N
N
A-85380 py5
N
N
CH3
A-84543 py2
A-98795-PH
Figure 1. Nicotinic ligands. Nicotinic ligands docked into (a4)2(ß2)3and (oc3)2(ß4)3 binding site.
89
Figure 2. a4ß2 pentamer model. Binding site region of the rat a4ß2 pentamer
model. In orange the résidus involved in formation of the (+)-side, in magenta
the résidus involved in formation of the (-)-side.
Figure 3. a3ß4 pentamer model. Binding site region of the rat a3ß4 pentamer
model. In orange the résidus involved in formation of the (+)-side, m magenta
the résidus involved in formation of the (-)-side.
90
The docking methodology was initially applied to the AChBP dimer modeled with
ACh in the cavity. AChBP binding site is located at the interface between two adjacent
momomcrs which behave, according to their residues, as the (+)- and the (-)-side of
the binding site region (12). Acetylcholine (Figures 4a) and (S)-nicotine docked into
the empty binding site of AChBP. Both ligands, which are natural ligands of AChBP,
docked very well into the binding site with the higher consensus score of 5. Figure 5b
shows that the oxygen carbonyl of acetylcholine establishes a H-bond with the O-H of
Tyr 192 (1.746 A0) of the (+)-side, whereas, the positively charged nitrogen is
stabilized by cation-Ti non-covalent interactions with the aromatic pocket made of
residues Trp 143, Tyr 89, 185, 192 of the (+)-side of the protein binding site. The
docking orientation of acetylcholine is in agreement with the position of the ACh
modeled in the binding site with the AChBP dimer (Bisson et al. 2003 submitted),
which is also shown in Figure 4b. The direction of the H-bond of the oxygen carbonyl
of acetylcholine changes but the orientation of the cation-7i interactions remains the
same.
a
91
Figure 4. AChBP model. Docking of acetylcholine into the binding site cavityof the AChBP dimer model, (a) docked acetylcholine in sticks and colored byatom type is put into comparison with the acetylcholine in sticks and colored in
red of the (AChBP + ACh) dimer model (Bisson et al. 2003 submitted).
Connolly Surface is created with SYBYL 6.8 (Tripos Inc., St. Louis, USA); (b)
binding site region of the AChBP dimer model. Docked ACh in white and modeled
ACh in yellow. In orange the residues of the (+)-side and in green the residues of
the (-)-side. Dashed lines represent H-bonds.
A series of nicotinic ligands were chosen (Figure 1) with binding affinity, calculated
with displacement binding studies with [3H]-cytisine, ranging from picomolar to
micromolar to cover ligand structural diversity and a broad range of binding affinity
for the rat a4ß2 neuronal nicotinic receptor (Table 1). Thus, the selection includes the
natural ligand acetylcholine, ligands from natural source like nicotine and epibatidine,
non-natural ligands like A-85380, A-84543, ABT-418 and derivatives from Abbott Co.
92
(USA), the epibatidine derivative dechloroepibatidine (20,21) and 33bMct, which was
synthesized in our group (Mu et al 2003 submitted).
The group of ligands were initially docked into the rat a4ß2 dimer as positive cations
and as neutral molecules. The docking with neutral molecules didn't work at all. All
the ligands were docked in their positively charged form and the consensus scores
were calculated for each docked compounds (Table 1). In this form the compound
docked within the binding site showing the importance of a positively charged nitrogen
in the structure as pharmacophoric group.
Two clusters were then identified. Cluster 1 and 2 are shown in Figure 5. Both
enantiomers of epibatidine and dechloroepibatidine docked in a similar orientation
(cluster 1) as shown in Figure 6a for (-)-epibatidine. Whereas (S)-85380, 33bMet and
acetylcholine docked in a different orientation (cluster 2) represented by (S)-85380 in
Figure 5.
Figure 5. Rat a4ß2 model. Docking of (-)-epibatidine and
(S)~A-85380 respectively in cluster 1 and 2. Dashed lines
represent H-bonds.
The common feature between the two clusters is the orientation of the positively
charged nitrogen, pointing toward the aromatic residues of the plus side (Trp 147 and
Tyr 188,195) and Trp 263 of the minus side. This underlines the importance of the
cation-7i interactions between the ligand and the receptor binding site. Cluster 1 and 2
revealed also the hydrogen bond between the pyridine nitrogen of the ligand and the
nitrogen of the side chain of Lys 285 (cluster 1 ) and of the main chain of Leu 327
93
(cluster 2) in the minus side of the ß2 subunit. It is important to observe that Leu 327
(119 of the ß2 subunit) by sequence homology is the residue Met 119 in the AChBP
involved in the hydrogen bond interaction with the carbonyl oxygen of acetylcholine
(12) (Figure 4b). This would confirm that the interaction between a H-bond acceptor
atom of the ligand and a H-bond donor atom of a residue of the receptor binding site is
an important driving force for affinity which is clearly represented by those docking
results. Moreover, in both clusters the carbonyl of Trp 147 of the plus side of a4
subunit makes a hydrogen bond with the NH of the positively charged nitrogen of the
epibatidine, dechloroepibatidine and (S)A-85380 stabilizing even more the docked
orientation of the ligands (Figure 5). Clusters 1 and 2, with the highest consensus score
found, involve the three mentioned nicotinic ligands which have, among the chosen
series, the highest affinity for the rat a4ß2 neuronal nicotinic subtype (Tabic 1 ).
The relationship between the shift of the pyridine nitrogen in (S)A-84543 from the
meta to the orto (S)A-84543o and para (S)A-84543p position and the binding affinity
was investigated. The three ligands (S)A~84543, (S)A-84543o, (S)A-84543p docked in
the same cluster 3 with lower consensus score than the ones found in cluster 1 and 2
(Table 1).
Figure 6. Rat cc4ß2 model. Docking of (S)-A-84543in cluster 3. Dashed lines represent H-bonds.
(S)A-84543 showed a higher consensus score than its derivatives, which is in
y^T
agreement with its higher binding affinity (Table 1 ). The positively charged nitrogen of
the ligands is directed toward the binding cavity but not as deep as in cluster 1 and 2
and this is the reason of the decrease of the number of positive interactions with the
binding site lowering the consensus score. The position of the nitrogen in the pyridine
ring seems to be critical for the affinity as it is shown in Figure 6. The nitrogen of
(S)A-84543 doesn't make any relevant contacts while the nitrogen of (S)A-84543p
would undergo the repulsion effect of the carbonyl oxygen of Cys 190 (3.284 Ä) in the
plus side of the a4 subunit if docked in the same way as (S)A-84543. This plays a big
role in the difference of affinity between these two ligands even both docked
orientations arc stabilized by the hydrogen bond between the NH of the positively
charged nitrogen and the oxygen carbonyl of Trp 147 in the plus side of the a4 subunit.
The nitrogen of (S)A-84543o makes an interaction with the side chain NH of Lys 285
(3.137 À) in the minus side of the ß2 subunit but the ligand looses the hydrogen bond
with Trp 147 present with the meta and orto derivatives. The depicted binding modes
allow us to draw a SAR related for this series of pyridine derivatives (Table 1).
(S)ABT-418 docked in the same cluster 3 with the positively charged nitrogen pointing
inside the binding cavity but this time the vicinity between the nitrogen and the oxygen
atoms in the aromatic ring of the ligand makes possible the hydrogen bond between the
aromatic nitrogen and the side chain NH of Lys 285 (2.222 Â) in the minus side of the
ß2 subunit, which is not present with (S)A-84543o and (S)A-84543p increasing its
consensus score. This is in agreement with the values of the binding affinity constant
which shows that (S)ABT-418 has affinity in the nanomolar range as (S)A-84543 and
much higher affinity than (S)A-84543o and (S)A-84543p (Table 1 ).
The effect on the affinity of the insertion of another nitrogen in the pyridine ring of the
ligand was studied too. The docking of (S)-2-pyraziny1~(A-84543py2) and (S)-5-
pyrimidinyl-(A-84543py5) derivatives of (S)-A-84543 gave the lowest consensus
score of 0 (cluster 4). Figure 7 clearly shows that the orientation of the docked
molecules is different. The positively charged nitrogen of the ligand is this time
pointing out of the binding cavity loosing the important cation-7i interactions to
estabilish a hydrogen bond with the carbonyl oxygen of Cys 190 in the plus side of the
a4 subunit (2.795 A for (S)A-84543py5 and 2.806 Ä for (S)A-84543py2). This
orientation is stabilized by the hydrophobic interactions between residues of the
binding site and the aromatic moiety of the molecule. This switching effect of those
95
docked ligands is probably due to the repulsions that would occur if the ligands would
bind with the same orientation as epibatidine. As a consequence the two ligands,
docked in the way mentioned before, loose the cation-7r interactions, lowering the
consensus score in agreement with lower affinity compared to (S)-A-84543 (Table 1 ).
This is in agreement with the demonstrated fact that the energy of interaction of a
hydrogen bond donor with a pyridine nitrogen is less favourable when a second
nitrogen is introduced into the aromatic ring, as in the case of diazine (23). The effect
on the orientation of the presence of a second nitrogen was also found with the (S)-3~
chloro-6-pyridazinyl-(A~84543py6Cl) which docked in same cluster 4 (Table 1).
Figure 7. Rat a4ß2 model. Docking of (S)-A-84543py2in cluster 4. In orange residues of (+)-side and in magenta
residues of (-)-side. Dashed lines represent H-bonds.
The best docking orientation of ligands having an oxygen bridge between the pyridine
and the aliphatic cycle moieties was observed decreasing the size of the aliphatic cycle
from aza octatabicycle to the azetidinc ring. Large and hindered aliphatic cycles make
difficult for the ligand to enter deeply into the binding cavity, loosing the possibility to
establish cation-rc interactions, which favorite the ligand binding to the receptor. This
explains the fact that by decreasing the size of aliphatic part of the ligand you get an
increase of binding affinity (24).
The ligand toxicity seems related with the docking orientation cluster 1 (Table 1). Both
cnatiomcrs of epibatidine and dechloroepibatidine docked well in cluster 1 and they
are all toxic compounds. Ligands which docked in the other high consensus score
96
orientation cluster 2 are non toxic compounds (Table 1). (S)ABT-418, which docked in
cluster 3 (Table 1), establishes similar interactions of epibatidine. Despite of this, the
orientation of (S)ABT-418 is different and less deep into the binding site cavity, as we
find in cluster 4, making loose cation-jr interactions. Thus, this might explain the
decreased binding affinity and the non toxicity of (S)ABT-418.
Regarding the a3ß4 binding site, both enantiomers of epibatidine and
dechloroepibatidine, which are potent agonists toward the ganglionic a3ß4 subtype
(10,25), docked well. Two clusters were identified. Cluster 1 and 2 are shown in
Figures 8.
Figure 8. Rat a3ß4 model. Docking of (-)-epibatidine
(white) and (+)-dechloroepibatidinc (yellow) into the
binding cavity of rat a3ß4 model. In orange residues
of (+)-side and in magenta residues of (-)-side. Dashed
lines represent H-bonds
In cluster 1, the positively charged nitrogen of (-)-epibatidine (Figure 8) is pointing
toward the aromatic pocket (Trp 147, 263 and Tyr 188, 195) revealing the importance
of cation-7t interactions between the ligand and the receptor binding site. The absence
of the H-bond interaction, between the side chain Lys 285 and the ligand pyridine
nitrogen, present in the a4ß2 binding cavity, is caused by the replacement of residue
Lys with lie. This is in agreement with the lower affinity of epibatidine toward the
ganglionic subtype (26) compared with the a4ß2. Moreover, the NH of the positively
charged nitrogen of both enantiomers of epibatidine and (-)-dechloroepibatidine
makes a H-bond with the oxygen carbonyl of Trp 147 of the plus side of a3 subunit.
97
The nitrogen of the pyridine ring establishes a H-bond with the hydrogen of the side
chain NH2 of Gin 325. For example, with (-)-epibatidine we found out a distances of
1.798 Â. Whereas, in cluster 2, the NH of the positively charged nitrogen of (+)-
dechloroepibatidine (Figure 8) makes still a H-bond with the oxygen carbonyl of Trp
147 but the pyridine ring is stabilized by the idrophobic interactions within the pocket
formed by the aromatic residues (Trp 147, 263 and Tyr 188, 195) inside the binding
cavity.
Acetylcholine and (S)-nicotine docked poorly into the binding cavity and this is in
agreement with their low potency agonism towards the the ganglionic a3ß4 subtype
(27).
(S)-ABT-418, (S)A-85380, (S)A-84543 and derivatives, that have a flexible ether
moiety and do not show toxicity, didn't dock into the binding cavity (Table 1). This
confirms that the presence of an oxygen between the pyridine and the aliphatic
moieties decreases the ligand affinity towards the ganglionic subtype with an increase
of selectivity toward the neuronal subtype.
The docking into the a3ß4 model of ligands which contain a cyclic ether bridging the
pyridine and the aliphatic ring such as the Abbott Co. (USA) ligand (S)A-98795, its
derivative (S)-A-98795-PH and 33bMet (Figure 1) was performed.
All three molecules docked well into the cavity with the high consensus score of 5
(Figure 9).
Figure 9. Rat o3ß4 model. Docking of(S)-A-98795
(white), (S)-A-98795-PH (green) and 33bMet (yellow).In orange residues of the (+)-side and in magenta residues
of the (-)-side. Dashed lines represent H-bonds.
Table t.
98
NAME es cluster es Ki(nM) toxicity
(Ref.) a4ß2 a4p2 a3p4 a4p2
(-)-Dechloroepibatidine 5 1 3 0.015ab toxic
(+)-Epibatidine 5 1 3 0.05 toxic
(-H)-Dechloroepibatidine 5 1 5 0.064a toxic
(-)-Epibatidine 5 1 3 0.073 toxic
33bMet 5 2 5 2.07 non toxic
(S)-A-85380 4 2 nd 0.05 non toxic
(S)-A-84543 3 3 nd 0.15 non toxic
acetylcholine 2 2 pd 7.6e non toxic
(S)-nicotine 2 pd 1 non toxic
(S)-A-84543 py5 0 4 nd 2.39
(S)-A-98795 5 2.7
(S)-ABT-418 3 3 nd 4 non toxic
(S)-A-84543 py2 0 4 nd 16.3
(S)-A-84543 6pyCl 1 4 nd 29
(S)-A-98795-PH 4 158
(S)-A-84543 o 2 3 nd 495
(S)-A-84543 p 2 3 nd 7914
nd, not docked in the cavity, pd, poorly docked in the cavity, a, brain homogenateswithout cerebellum, b, IC50 data, c, with [3H]acetylcholine
33bMet was proved to behave as an agonist towards the ganglionic a3ß4 subtype
(Bisson et al. 2003 submitted). The docking orientation of (S)A-98795, (S)-A-
99
98795-PH and 33bMet is similar to (+)-dechloroepibatidine (cluster 2). In fact, the
positively charged nitrogen is in all cases pointing away from the aromatic surrounding
of the plus side of the a subunit making this time, with its hydrogen, a H-bond
interaction with the oxygen of the carbonyl of the residue Gin 325 of the minus side of
the ß subunit. The H-bond distances with (S)A-98795, (S)-A-98795-PH and 33bMet
found are respectively 1.989 Â, 2.014 Â and 1.933 A. The aromatic nitrogen of the
ligand (S)-A-98795 seems to not play such a big role in the binding affinity to this
ganglionic subtype.
The docking orientation of the ligands into the a3ß4 model binding site (cluster 1 and
2), are related with ligand agonism potency. Ligands with flexible ether moieties do
not dock. Whereas, the effect of sterically constrained and planar C-O-C and C-C-C
angle, as with ligand (S)-A-98795 and 33bMet closed to the epibatidine structure,
improves the docking of the ligand into the binding cavity and the affinity towards the
subtype receptor.
Conclusions
A molecular docking study was performed on the dimer model of the geometrically
and stereochemically refined rat neuronal a4ß2 and ganglionic a3ß4 nicotinic subtype
receptor.
The compounds chosen for the docking screening are known nicotinic ligands. They
all have the right structural features for binding to nicotinic receptor like a positively
charged nitrogen, an electrondonor atom like the pyridine nitrogen capable of
hydrogen bonding and a centre of a hydrophobic area generally occupied by aliphatic
cycles. The docking results and the consensus scores of the docked molecules
confirmed and gave a satisfactory structure-activity relationship explaining the putative
binding modes of the ligands. Moreover, regarding the docking on the a4ß2 model the
consensus score evaluation showed significant correlation with the differences in
binding affinities, while, single scoring functions did not allow any discrimination.
Interesting results were found regarding the investigation of the selectivity of the
ligands between central neuronal and peripheral ganglionic nicotinic subtypes. From
all ligand examined both enantiomers of epibatidine and the derivative
dechloroepibatidine are the only one docking with high consensus scores in both
binding cavities. This is in agreement with the experimental values on a4ß2 and the
100
peripheral side effects, linked to a3ß4, obtained during their clinical use. Two
important clusters of docking (1-2) are found in the a4ß2 model involving high affinity
(picomolar range) ligands like epibatidine, dechloroepibatidine and (S)-A-85380 and
acetylcholine and 33bMet. The orientation of the docked molecules underline the
importance of cation-^ interactions between the positively charged nitrogen of the
ligand and the aromatic side chains of residues at the plus side of the a4 subunit and of
the hydrogen bond contacts between the pyridine nitrogen or oxygen carbonyl of the
ligand and the backbone NH-of residues mainly of the minus side of the
complementary ß2 subunit. The type of ligands which docked in cluster 1 and 2 were
respectively the clinically toxic epibatidine and dechloroepibatidine and the clinically
not toxic (S)A-85380, 33bMet and acetylcholine. Regarding the docking of the ligands
into the a3ß4 model binding site, which seems to be highly correlated with ligand
agonism potency, the results revealed two important factors. The presence of the
oxygen atom between the pyridine ring and the aliphatic moiety plays an important
role in abolishing affinity to the ganglionic receptor, represented by the impossibility
of finding a binding orientation within the binding site. Thus, this would increase the
subtype selectivity toward the neuronal receptor. This indicates that the insertion of the
oxygen would decrease the toxicity of the ligands by reducing the affinity towards
peripheral nicotinic subtypes. If the flexibility around the C-O-C or C-C-C- angles is
constrained, as with ligand (S)-A-98795 and 33bMet, the chance to dock into the a3ß4
binding cavity increases showing that the tridimensional structure of epibatidine is a
good template for ligands affine to the ganglionic receptor but with low subtype
selectivity.
In general the ranking consensus scores of the docked molecules showed a correlation
with the inhibition constant (Ki) values taken from literature (see Table 1).
The results obtained from the docking investigations showed that those models are
useful to improve subtype selectivity by designing compounds with high affinity
towards the neuronal a4ß2 subtype (preferably cluster 2 mode) and with enough
flexibility to not bind to the ganglionic a3ß4 subtype. This would improve the clinical
and safety profile oftherapeutic and diagnostic applications in neurological diseases.
101
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104
CHAPTER 6
Conclusions and Outlook
In recent years, it has become clear that the neuronal nicotinic acetylcholine receptors
(nAChRs), ligand gated ionic channels, may play a role in a variety of diseases,
including Alzheimer's disease (AD). The alkaloid epibatidine (EPB), originally
isolated from the skin of the Ecuadorian poison frog, Epipedobates tricolor, by Daly
and co-workers, is a very specific agonist of the neuronal nAChR. However, the
toxicity of this molecule prevents its therapeutic and even diagnostic use in clinical
medicine.
In the present project thesis, to develop new ligands selective for distinct nAChR
subtypes with a satisfactory safety profile, new analogues of epibatidine and cytisine
were synthesised and investigated by in vitro binding and electrophysiology studies.
These compounds have all the key pharmacophore characteristics important for the
binding to the nicotinic receptor but the interatomic distances and angles between these
structural features change from ligand to ligand influencing the receptor binding and as
a consequence the subtype selectivity.
The inhibition constants (Kx) of the novel synthesised ligands towards the 0^2 and an
neuronal nicotinic receptors were estimated using the inhibition competition binding
assay method respectively with [3H]-cytisine and [125I]-a-bungarotoxin. Compound
(±)-33bMet (represented below) showed high affinity (Ki = 2 nM) and brain subtype
selectivity (a$ila-i affinity ratio >100).
105
Voltage clamp electrophysiology was performed with these compounds on the
neuronal a4ß2 and a7 and on the ganglionic a3ß4 nAChRs reconstituted in Xenopus
oocytes.
(±)-33bMet exhibited partial agonism towards neuronal o^2 nAChR subtype and
relatively low toxicity on mice (LD50 > 0.5 mg/kg body weight) has been
demonstrated.
Thus, the results obtained revealed that, within the series, the synthesized compound
(±)-33bMet is suitable for animal PET investigations. It will be then labeled with nC
and 18F and injected intravenously in rats to investigate and develop a new series of
positron emission tomography (PET) tracers for imaging neuronal nAChRs.
The crystalline structure of the acetylcholine-binding protein (AChBP) has been
recently resolved. This opened possibilities to model different nAChR subtypes using
AChBP as template for the extracellular N-terminal domain of the nAChR subunits.
In the present project thesis, the high homology between the different nicotinic
subunits from different species has allowed us to build the models of the extracellular
domain of the rat and human (04)2^2)3 and (a7)5 nicotinic receptor subtypes which are
the most present in the brain. These models were for the first time successfully
assessed by Molecular Dynamic and show conformational stability over time. Hence,
they may be useful, with longer dynamics, to investigate the overall tridimensional
movements of the pentamer with or without the presence of the ligand in order to
understand the allosterism between the different binding sites and the kinetic of the
extracellualar domain ofthe rat and human receptor subtypes.
A molecular docking study was performed on the dimer model of the geometrically
and stereochemically refined rat neuronal 0^2 and ganglionic a3ß4 nicotinic subtype
receptor. The compounds chosen for the docking screening are known nicotinic
ligands. The docking results and the consensus scores of the docked molecules
confirmed and gave a satisfactory structure-activity relationship explaining the putative
binding modes of the ligands. Interesting results were found regarding the
investigation of the selectivity of the ligands between central neuronal and peripheral
ganglionic nicotinic subtypes. From all ligand examined both enantiomers of
epibatidine and the derivative dechloroepibatidine (see figure below) are the only one
docking with high consensus scores in both binding cavities. This is in agreement with
the experimental values on 0^2 and the peripheral side effects, linked to a3ß4, obtained
106
during their clinical use. Two important clusters of docking (1-2) are found in the o$2
model involving high affinity (picomolar range) ligands like epibatidine,
dechloroepibatidine and (S)-A-85380 and acetylcholine and 33bMet (see figure
below). The orientation of the docked molecules underline the importance of cation-^
interactions between the positively charged nitrogen of the ligand and the aromatic
side chains of residues at the plus side of the 04 subunit and of the hydrogen bond
contacts between the pyridine nitrogen or oxygen carbonyl ofthe ligand and the
I NMe
sr
33bMet
^t "X.Dechloroepibatidine
A-85380
Acetylcholine
A-98795
backbone NH-of residues mainly of the minus side of the complementary ß2 subunit.
The type of ligands which docked in cluster 1 and 2 were respectively the clinically
toxic epibatidine and dechloroepibatidine and the clinically not toxic (S)A-85380,
33bMet and acetylcholine (see figure above). Regarding the docking of the ligands into
the a3ß4 model binding site, which seems to be highly correlated with ligand agonism
potency, the results revealed two important factors. The presence of the oxygen atom
between the pyridine ring and the aliphatic moiety plays an important role in
abolishing affinity to the ganglionic receptor, represented by the impossibility of
finding a binding orientation within the binding site. Thus, this would increase the
subtype selectivity toward the neuronal receptor. This indicates that the insertion of the
oxygen would decrease the toxicity of the ligands by reducing the affinity towards
107
peripheral nicotinic subtypes. If the flexibility around the C-O-C or C-C-C- angles is
constrained,, as with ligand (S)-A-98795 and 33bMet (see figure above), the chance to
dock into the a3ß4 binding cavity increases showing that the tridimensional structure of
epibatidine is a good template for ligands affine to the ganglionic receptor but with low
subtype selectivity.
The results obtained from the docking investigations showed that those models are
useful to improve subtype selectivity by designing compounds with high affinity
towards the neuronal 04ß2 subtype (preferably cluster 2 mode) and with enough
flexibility to not bind to the ganglionic a3ß4 subtype. This would improve the clinical
and safety profile oftherapeutic and diagnostic applications in neurological diseases.
Rat and human subtype data will be put in comparison to study the relationship
between different species and to confirm the common origin in the evolutionary tree of
nicotinic ligand- gated ionic channels.
108
PUBLICATIONS AND PRESENTATIONS
Papers:
L. Mu, W.H. Bisson, G. Westera, A. Schibig, C. Wirz, P.A. Schubiger. Synthesis and
binding studies evaluation of epibatidine analogues as novel ligands for the Nicotinic
Acetylcholine Receptors (submitted to J. Med. Chem).
W.H. Bisson, G. Westera, S. Bertrand, L. Mu, A. Schibig, J. Rouden, D. Bertrand and
P. A. Schubiger. Affinity and electrophysiological evaluation of epibatidine and
cytisine analogues as novel ligands for the Nicotinic Acetylcholine Receptors
(submitted to Chem and Biol).
W.H. Bisson, G. Westera, P.A. Schubiger, and L. Scapozza. Homology Modeling,
Dynamics of Rat and Human Extracellular Domain of a4ß2 and a7 Neuronal
Nicotinic Acetylcholine Receptor Subtypes (submitted to J. ofComp.-aided Mol Des).
W.H. Bisson, G. Westera, L. Mu, P.A. Schubiger, and L. Scapozza. Molecular docking
to investigate ligand selectivity for the acetylcholine binding site of the rat a4ß2 and
a3ß4 nicotinic subtypes (submitted to J. Med. Chem).
Poster and Oral Presentations:
W.H. Bisson. Synthesis and Characterization of new ligands for the neuronal nicotinic
acetylcholine receptor. Doktorandentag Pharmazie-ETH, Zürich, Switzerland, April
2002
W.H. Bisson, L. Mu, P.A. Schubiger, G. Westera. Synthesis and Characterization of
new ligands for the neuronal nicotinic acetylcholine receptor. 3r Annual Congress of
the Swiss Society ofNuclear Medicine, Aarau, Switzerland, June 2002
109
W.H. Bisson, L. Mu, S. Bertrand, J. Rouden, A. Buck, P.A. Schubiger, D. Bertrand, G.
Westera. Synthesis and Characterization of new ligands for the neuronal nicotinic
acetylcholine receptor. ZNZ-Symposium 2002, Zurich, Switzerland, October 2002
W.H. Bisson, L. Mu, S. Bertrand, J. Rouden, A. Buck, P.A. Schubiger, D. Bertrand, G.
Westera. Synthesis and Characterization of new ligands for the neuronal nicotinic
acetylcholine receptor. 32nd Annual Meeting of the Societyfor Neuroscience, Orlando,
FL, USA, November 2002
W.H. Bisson, G. Westera, L. Mu, P.A. Schubiger, and L. Scapozza. Stracture-activity
relationship to investigate ligand selectivity for the acetylcholine binding site of the rat
a4ß2 and a3ß4 nicotinic subtypes. 226'"' National Meeting of the American Chemical
Society, New York, NY, USA, September 2003
110
CURRICULUM VITAE
Name
Born
Bisson William Henry
28.04.74 in Gent (B)
Nationality Italian
1980-1988 Primary and Secondary School in Treviso (1)
1988-1992 High School at the "European School" in Brussels (B)
1992-1999 Master Degree in Chemistry, University of Padua (I).
Dissertation under Prof. C. Toniolo.
Subject: Towards the identification of a 3jo-helical peptide
conformation by means ofNMR spectroscopy.
1999-2000 Research Stage, DSM Research, Geleen (NL)
Supervision: Dr. Q. Broxterman and Dr. B. Kaptein
Subject: Testing ofnew bio-resolution concepts and "large
scale" preparation of an enantiopure a,a-disubstituted
amino acid.
2000-2003 PhD Degree, CHAB-ETH, Zürich, Switzerland.
Dissertation under Prof. Dr. P.A. Schubiger.
Subject: Novel ligands for the neuronal nAChR: synthesis,
in vitro characterizations and binding models.
PUBLICATIONS
M. Crisma, W.H. Bisson, F. Formaggio, Q.B. Broxterman, C. Toniolo. Factors
governing 3io-helix vs a-helix formation in peptides: percentage of Ca-tetrasubstituted
a-amino acid residues and sequence dependence. Biopol 2002, 64, 236-245.
Ill
A. Dehner, E. Planker, G. Gemmecker, Q.B. Broxterman, W.H. Bisson, F. Formaggio,
M. Crisma, C. Toniolo and H. Kessler. Solution structure, dimerization, and dynamics
of a lipophilic a/3io-helical, Ca-methylated peptide. Implications for folding of
membrane proteins. J. Am. Chem. Soc. 2001, 123, 6678-6686.
**cover illustration ofthe J. ofPept. Res. 2001, 58(1).
H. Schedel, P.J.L.M. Quaedflieg, Q.B. Broxterman, W.H. Bisson, A.L.L. Duchateau,
I.C.H. Maes, R. Herzschuh and K. Burger. Synthesis of (R)-4,4,4-trifluoro-2-
mercaptobutyric acid. Tetrahedron Asymmetry 2000, 11,2125-2131.
112
ACKNOWLEDGMENTS
I would like to thank Prof. Dr. P. August Schubiger for giving me the opportunity to
accomplish my doctoral thesis in his group, for introducing me in the field of
radiopharmacy and for his capacity to hear.
I would like to thank Prof. Dr. G. Folkers for agreeing to be my coreferent in the
present thesis and for his way to deal with people.
My sincere thanks are due to Dr. Gerrit Westera for his supervision, his constructive
criticism and his support.
I am very grateful to Prof. Dr. L. Scapozza for his supervision, his sympathy and for
introducing me in the field of computational chemistry.
I would like to thank Prof. Dr. D. Bertrand and his wife Sonia from the University of
Geneva for their kidness, their experience and collaboration in the field of
electrophysiology.
I am very grateful to Dr. L. Mu and D. K. Drandarov for their experience in the field of
organic chemistry, their support and friendship.
Many thanks are further due to Dr. M. Jakusch and Dr. M. Honer for their experience
respectively in the field of computational chemistry and biochemistry.
I would like to thank the students Arianna, Ivan, Christa and Anita for their works
which are part of the thesis.
My sincere thanks are due to Pavel, Mine, Maria, Katia, Thomas, Patrick, Ingrid and
all Scapozza-Folkers group for their friendship and for the nice atmosphere around the
M-Stock.
113
I would like to thank Daniel, Sem, Michèle, Regis, Fred, Christoph, El Mache, Junior,
Niko, Maka, Anass, Metin, Beat, Mauro, Michelle, Enzo, Tchuge, Arturo and all
friends for the nice moments after work passed together.
My special thanks are due to my family and Jeannine for their constant support and
bearing me at any time.
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