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Health Sciences 1I06
The UNSIN Report
Thursday April 5, 2012
Group Name:
Group Members:
Team Indra
Patrina Cheung,
Christopher Chung,
Katelyn Colwell,
Yasmeen Mansoor,
Laura Morrison,
Sinthuha Sivananth,
Andrew Webster,
Brendan Wong,
Yezarni Wynn
Part 1: Abstract
Wrath is the physical component of anger in which an individual outwardly
displays extreme aggression, inflicting harm on others or themselves. Many complex
biological pathways are involved in the regulation of aggression - the most prominent being serotonergic. In many studies, intense aggression has been linked to low
postsynaptic 5-HT1A receptor activity in the prefrontal cortex (PFC). More specifically, the orbitofrontal cortex (OFC - a region of the PFC) is believed to be involved in
inhibiting aggression. Thus, Indra Pharmaceuticals has decided to focus on increasing 5-HT1A postsynaptic activity in the OFC as a key intervention target for reducing
aggression.
ChillPil will be a safe, highly efficacious drug capable of treating a large population of dangerously aggressive people who possess some form of decreased 5-HT1A
activity in the OFC. Its primary active ingredient will be developed from 2-(2H-1,4benzoxazin-3-ylamino)benzonitrile (BZN), which is a positive allosteric modulator
that Indra Pharmaceuticals has designed to increase receptor affinity for endogenous
serotonin at postsynaptic 5-HT1A receptors. In vitro experimentation demonstrated
that BZN selectively and effectively increases postsynaptic 5-HT1A receptor activity. Finally, proof-of-concept behavioural tests conducted on mice administered with
BZN resulted in notably reduced aggressive behaviour.
Abbreviations: PS 5-HT1A - postsynaptic 5-HT1A receptor; OFC - orbitofrontal
cortex; PFC - prefrontal cortex; BZN - 2-(2H-1,4-benzoxazin-3-ylamino)benzonitrile; SSRI - selective serotonin reuptake inhibitor; GPCR - G-protein coupled receptor; PKA - protein kinase A; FLIPR - Fluorometric Imaging Plate Reader; FlCRhR
- Cyclic AMP Fluorosensor.
Part 2: Background Information
An Introduction to Wrath
Dopaminergic
High levels of dopamine positively induce
aggression, and high D2 receptor activity
in particular has been implicated [1].
Further proof stems from the historical
use of D2 receptor antagonists such as
haloperidol to treat aggressive behaviours
in psychotic patients [1,3-4]. Additionally,
single point mutations leading to
decreased activity of dopamine regulating
enzymes have been correlated with
increased aggression [5]. One example
Molecular Pathways Involved is the single base pair replacement in
the DARPP-32 gene (for the dopaminein Wrath
regulating phosphoprotein) which was
correlated with higher ANGER scores
GABAergic
and found in most patients with bipolar
(conditions
Increasing the synaptic concentration disorder and schizophrenia
[6]
linked
to
aggression)
.
However,
of gamma-Aminobutyric acid (GABA)
such
mutations
are
only
seen
in
a small
has been shown to reduce aggression
population.
Also,
the
most
historically
in both rat and human tests, suggesting
that those who are aggressive exhibit successful method of reducing aggression
lower GABAergic activity [1,2)]. However, a through D2 receptor antagonism causes
definitive source of decreased GABAergic adverse side effects such as locomotion
activity in aggressive individuals is still impairment and sedation, thus implying
unknown. Research suggests that in the that intervention in[1,3]the dopaminergic
neurobiological mechanism for escalated pathway is not ideal .
aggressive behaviour, GABAergic activity
is modulated by serotonergic neurons Noradrenergic
[1]
. Thus, the coinciding role of serotonin
in GABA modulation poses potential Enhanced norepinephrine activity via
receptors
observed
complications in choosing this pathway beta-adrenergic
in
the
brainstem
has
been
associated
as a locus for intervention.
with aggressive behaviour [7]. However,
Anger is a natural emotion that the entire
human population experiences. Wrath, on
the other hand, is a physical component
of extreme anger, in which an individual
displays intense aggression. While a
number of external factors play a role in
evoking aggression, there are also certain
neurotransmitter and hormone pathways
(Fig. 1) that when disrupted, can result in
violent behaviour.
FIGURE 1: Potential loci for intervention in wrath. Each row outlines the various pathway levels that
were considered, whereas the bolded terms specify the route taken in order to cure wrath.
in animal studies, both agonists and
antagonists to adrenergic receptors were
observed to reduce aggression. As such,
there is conflicting evidence of a positive
relationship for norepinephrine and
aggression [3].
Cortisol and Testosterone Pathway
Studies indicate that an imbalance in the
endogenous levels of testosterone and
cortisol can lead to decreased activity
in the medial orbitofrontal cortex,
resulting in aggression [8,9]. However,
the sole manipulation of testosterone
levels in humans does not seem to
produce a consistent effect on wrathful
behaviour [10]. The triple balance model
of emotion is the best explanation for
the roles of cortisol and testosterone in
anger modulation, incorporating various
social and psychological factors[11-13].
However, it does not readily give a strong
explanation for extreme aggression
such as wrath. Literature supporting
the role of testosterone and cortisol in
anger is sparse, therefore this means of
intervention is unfavourable.
Serotonin
Extensive research has suggested an
inverse relationship between serotonergic
activity and wrathful behaviour [14].
This correlation has been displayed
in human subjects with personality
disorders, violent offenders, patients with
depression and anger attacks, as well as
nonhuman primates [15,16]. This affirms
that various forms of aggression stem
from decreased responsiveness of the
serotonergic system. Thus, this pathway
is optimal for drug intervention.
Brain Region Pathways
Involved in Wrath
Studies have shown that the activity of
serotonin in many regions within the
prefrontal cortex (PFC) plays a notable
role in modulating anger [1,17]. In the
non-human primate model, aggressive
behaviour is evoked by visual and auditory
information, which is sent to both the
orbitofrontal cortex (OFC - a region of the
PFC) and the medial amygdala (Fig. 2).
The OFC primarily inhibits the function of
the amygdala, repressing the aggressioninducing signal pathway [18]. Damage to
the OFC has been shown to contribute
to aggressive behaviour, suggesting
that those who are prone to aggression
possess decreased OFC activity [18]. Thus,
enhancing serotonergic activity in the
OFC is a viable route for suppressing
aggression.
FIGURE 2: The brain pathways involved in
aggression of non-human primates. In this
pathway, external stimuli are processed into visual
and auditory information, which are then sent to
both the OFC and the medial amygdala. The medial
amygdala proceeds to transmit signals to other areas
of the brain such as the anterior hypothalamic area
and the bed nucleus of the stria terminalis, which
activate the periaqueductal grey in promoting
aggression [3].
.
Types of Serotonin Receptors
The serotonin receptors most abundant
in the PFC (which contains the OFC) are
the 5-HT1A, 5-HT2A and 5-HT3A receptors.
However, it was found that 5-HT1A
activation was dominant in producing
the inhibitory effects of PFC neurons,
including the suppression of aggressive
behaviour [19]. In particular, both healthy
and personality disordered individuals
with high levels of aggression showed
reduced 5-HT1A receptor function [20].
Also, 5-HT3A receptors are exclusively
found on superficial cortical layers,
whereas 5-HT1A and 5-HT2A are expressed
in deeper layers of the PFC [19]. These
layers play a greater functional role in
controlling emotions, and decreased
activity of these areas results in mood
disorders and schizophrenia [21]. Thus,
5-HT1A receptors have a dominant role
over 5-HT2A and 5-HT3A receptors in PFCmediated control over aggression due to
their strong suppressive capabilities and
high density in deeper layers of the PFC.
and rapid blood pressure due to excessive
serotonin [14, 25].
Post-Synaptic vs. Pre-Synaptic
5-HT1A Receptors
Additionally, mutations of the MAO-A and
Postsynaptic 5-HT1A (PS 5-HT1A) has been
shown to serve a more predominant role
in regulating aggression versus 5-HT1A
autoreceptors.
These
autoreceptors
regulate synaptic serotonin concentrations
and as such, their activity affects a
number of postsynaptic serotonergic
responses that are not necessarily
involved in aggression [22]. Thus, the PS
5-HT1A receptor is more favourable for
intervention.
tryptophan hydroxylase genes are capable
of interfering with PS 5-HT1A activity
[6, 26]
. To address such issues, the ideal
treatment would be gene therapy. This
would treat both excessive and deficient
serotonin levels while displaying minimal
side effects. However, such mutations are
not prevalent, reducing the scope of this
method to only a miniscule population [6].
After considering the disadvantages of
increasing agonist concentration and
gene therapy, allosteric modulation is the
Research suggests that reduced PS 5-HT1A optimum method for drug intervention.
receptor function can result from low Allosteric modulation has numerous
endogenous serotonin, low serotonin advantages over orthosteric receptor
binding affinity, low receptor expression, agonism, including greater selectivity of
gene mutations, desensitization or G-protein coupled receptors (and thus
decreased function of the receptor [20, 21]. reduced side effects), and improved
[27,28]
responses
Hence, possible methods of intervention pharmacological
.
include increasing synaptic serotonin or Additionally, allosteric modulation can
agonist concentrations, gene therapy for even overcome effects of natural negative
mutations and allosteric modulation of allosteric modulators such as zinc [29].
PS 5-HT1A.
Allosteric modulation is an ideal approach
as it allows the body to retain its natural
control over receptor activation, while
Methods to Alter 5-HT1A
allowing for control over the intensity of
Activity
the response.
A common approach to enhancing
serotonergic receptor activity involves Target Population
increasing
synaptic
serotonin
concentrations,
either
via
the Through allosteric modulation of the PS
administration of exogenous serotonin or 5-HT1A receptor, Indra Pharmaceuticals
autoreceptor antagonism. However, this will address a vast, wrathful population
is the logic behind most antidepressant whose behaviour can result from
and antianxiety medications, which have reduced 5-HT1A receptor function (e.g.
a plethora of adverse side effects [23]. One desensitization), low agonist binding
inherent risk in increasing synaptic levels affinity, or low receptor expression. These
of any neurotransmitter is the risk of it factors all contribute to decreased 5-HT1A
binding to another receptor in the central activity in the OFC, therefore resulting in
nervous system. For example, Prozac, aggressive behaviour.
the most widely prescribed selective
serotonin reuptake inhibitor (SSRI) also
antagonizes cholinergic receptors giving
rise to blurred vision, constipation, dry
mouth and drowsiness [24]. In addition,
the overactivation of postsynaptic
serotonergic receptors by SSRIs can lead
to down-regulation in the long run [24].
Furthermore, SSRIs have been known to
interact with forty other drugs to cause
serotonin syndrome, characterized by
restlessness, fever, increased heartbeat
Part 3: Accomplishments, Plans, JYP
Creation of the Ligand
Functional Readout Tests
BZN [ 2-(2H-1,4-benzoxazin-3-ylamino)
benzonitrile] is a high-efficacy allosteric
modulator of PS 5-HT1A receptors. Based
on comprehensive in-silico research
into the dynamic equilibrium and MD
trajectory inter-equilibrium states of PS
5-HT1A, two positive allosteric binding sites
were identified (key interacting residues
Trp-364 (TM7) and Glu-147 (TM3)) on the
receptor [30-36]. These have been identified
as organic solvent fragment accessible, as
well as energetically favourable for binding
[37-39]
. Most importantly, when bound to,
these sites conformationally modulate
PS 5-HT1A to increase the likelihood
of endogenous serotonin successfully
activating the receptor (by reducing the
Koff value of serotonin interaction with the
p1 pocket of PS 5-HT1A) and optimizing
the steric conditions required for
G-protein coupling to PS 5-HT1A (through
modulation of the iono-lock pathway)
[36]
. After identifying these target sites,
we performed successive virtual highthroughput screenings of the ZINC clean
lead-like library to identify non-patented
lead candidates [40,41]. The clean lead-like
library is a database of core functional
structures that can act as the foundation
for more complex drug-like compounds
[42]
. In this sense, these leads are ideal
in that they can be easily modified and
added onto to enhance their selectivity or
functional properties. BZN (Fig. 3) was
identified as the top dock-scoring lead
compound that passes Lipinski’s Rule of
Five, can pass through the blood-brain
barrier, and also does not bind efficaciously
to other regions of
5-HT1A [40-42].
Objective 1: Testing Ability to
Modulate the 5-HT1A receptor
FIGURE 3: 2-(2H-1,4benzoxazin-3-ylamino)
benzonitrile
A series of tests were done to determine
whether BZN efficaciously increases
activation of the PS 5-HT1A receptor.
Normally, once an agonist binds to the
receptor, the G-protein subunit of the
receptor inhibits adenylyl cyclase and
subsequently decreases intracellular
cyclic adenosine monophosphate (cAMP).
cAMP phosphorylates protein kinase
A (PKA), resulting in PKA dissociation
[43]
. Thus, to quantify positive PS 5-HT1A
intracellular response, PKA dissociation
was measured using FlCRhR analysis
(Invitrogen) [44-47]. Tests were conducted
in vitro on neuronal PS 5-HT1A receptorexpressing cell lines (EMD Millipore) [48].
Five cell systems were pretreated with
the substrates indicated in the legend of
Figure 4. This was followed by the equal
administration of forskolin (an adenylyl
cyclase inducer) and serotonin (the
activating ligand of 5-HT1A) to each system
[49,50]
. Positive and negative controls (H89
and WAY-100635, respectively) were
used to compare the efficacy of BZN
against basal levels [49, 51]. Zinc, a negative
allosteric modulator, was used with BZN
to determine the affinity of BZN to 5-HT1A
[29]
.
Results
Cells pretreated with BZN displayed
notably decreased levels of PKA
dissociation compared to unmodulated
receptors in the presence of equal doses of
serotonin and forskolin. The tests detail
BZN’s ability to increase 5-HT1A activity
See Appendix (Part to a greater degree than the positive
3.5) for full details of control, illustrating its high efficacy.
Also, results implicate that the allosteric
ligand design.
mechanism of BZN was more prominent
than that of zinc, explaining the similar
PKA ratio curves between systems 4 and
5. This supports that BZN has a powerful
allosteric modulatory effect on 5-HT1A
receptors that increases the receptor
affinity for 5-HT.
Figure 4: Neuronal fluorescence ratio
vs. time. This graph shows fluorescence
ratio over time. FlCRhR measures the
dissociation of PKA using a modified
FRET system. Rhodamine and fluorescein
are fluorophores for the regulatory and
catalytic subunits of PKA. Without cAMP
phosphorylation, rhodamine emission is
higher since it is co-activated. However,
after cAMP activates PKA, fluorescein is
separated from the rhodamine fluorophore
and thus, the fluorescence ratio increases.
At t=0, 25 x 10-5M forskolin and 10 x 105
M serotonin were administered to each
system. Test 4 indicates that BZN amplifies
the serotonin induced decrease of PKA
ratios by 80.7% compared to test 1. (45,46)
Figure 5: Dose-dependent response of
PKA to BZN. The same method from
objective 1 was used. Each cell system
was pretreated with BZN at various
concentrations indicated by the legend.
At t=0, 25 x 10-5M forskolin and 10 x
10-5M serotonin were administered to
each system. At increasing concentrations, BZN was able to further inhibit
the dissociation of PKA. The results
draw a negative relationship between
BZN and fluorescence ratio and therefore, cAMP. (52)
Objective 2: Testing Dose Response
Efficacy
Objective 3: Testing for GPCR
Selectivity and Effects on Other
Receptors
To confirm the direct role of BZN, a
dose-response test was conducted using
varying concentrations of BZN but the
same concentrations of forskolin and
serotonin as the previous test.
The method of testing involved the use
of FLIPR calcium assay kits (Molecular
Devices) to measure intracellular calcium
level changes in response to BZN and
respective agonists [53,54]. Cells that
expressed the following receptor subtypes
Results
were purchased (Millipore): 5-HT (1A, 1B,
2A, 3A), dopamine D2, alpha (1,2) and beta
Results (Fig. 5) showed that there was (1, 2, 3) adrenergic receptors, and GABA
a proportional decrease in fluorescence B receptors [48]. These receptors were
ratio as BZN concentrations were selected due to their G-protein coupled
increased. Thus, it was drawn that there mechanism and possibility of them
is a direct positive correlation between interfering in the serotonergic or wrath
cAMP inhibition and BZN concentrations pathways in the OFC. Preparations were
[52]
.
done according to previous procedures
[55]
.
Test A) Primarily, each expressed
cell line was administered a constant
concentration of BZN and placed into a
Figure 6: FLIPR-based assay results testing BZN’s ability to elicit a response on various
receptors. Shown are results indicating that BZN administered as an agonist does not elicit a
response on any receptor subtypes (BZN+all receptors). Additionally, the change in response from
agonist alone to agonist+BZN in all non-5-HT1A receptors is minimal, indicating that in the presence
of BZN, the only positive effect is seen on the target 5-HT1A receptor. The units on the y-axis are delta
fluorescence units or change in fluorescence against log concentration of agonist. [56]
FLIPR to monitor cell fluorescence before
and after the addition of BZN. This was
done to test the ability of BZN to elicit a
response on the aforementioned receptors
without their respective agonists.
Test B) In the first round, each cell line was
administered increasing concentrations
of its respective agonist (e.g. dopamine
to dopaminergic receptors) and the
fluorescence was observed with a FLIPR
assay. This served the purpose of a
baseline dose-response curve to compare
to agonist+BZN. In the second round, the
cells were pretreated with 10-10M BZN
and exposed to increasing concentrations
of their respective agonist. Change in
fluorescence was again observed with a
FLIPR assay. The dose-response curves
were constructed using the maximal
response taken from the various
concentrations [56].
Results
The results from test A and B can be seen
in Figure 6. For test A, no response was
seen by adding BZN alone in any of the
receptors and as such one curve was made
to indicate the average response. This is
labelled “BZN+all receptors”. For test B,
for clarity purposes, the results for each
neurotransmitter pathway were averaged
into one fluorescence curve. For example,
the curve showing “dopaminergic
receptors” was the average fluorescence
change observed across all dopamine
receptor subtypes.
When the cells were administered only
BZN, no response was seen, indicating
that BZN does not possess agonistic
properties. A second test was conducted
to confirm that BZN was not an antagonist
or an allosteric modulator on non-5HT1A
receptors. When the cells were pretreated
with BZN and administered their
respective agonists, the dose-response
curves were identical to administration of
just the agonist. The only exception was
for 5-HT1A, where there was an increase in
response -- as should be the case for BZN.
From the above results, it can be concluded
that BZN selectively, allosterically binds
to and positively modulates the PS 5-HT1A
receptor.
Figure 7: Effects of Drug Administration on Social
Dominance. Measuring percentage of dominance was
based on previous social dominance studies [59,60]. Percentages were based on trials in which the occurrence
of dominance upon drug intake was observed in the
dominant mouse (identified from baseline tests). BZN
was shown to reduce dominance by 24.0%, compared
from 16.0% from serotonin administration.
Objective 4: Testing for
functionality in vivo
In vivo testing involved monitoring
changes in aggression invoked by BZN
in mice, a common animal model for
drug testing. Mice are often used as
test subjects due to their comparable
anatomy and physiology to that of
humans [57]. All test mice involved were
acclimated in a vivarium for one week
prior to experimentation, under identical
conditions [58].The social dominance [5861]
and shock-induced aggression tests
[58, 62]
were used to measure the changes
in aggression before and after the
administration of BZN.
Results
Test A) Social Dominance Test:
Dominance
is
representative
of
aggression. In baseline testing, one mouse
dominated 34 out of 50 trials. Then, BZN
was administered to this dominating
mouse via cannula (Strategic Applications
Inc.) to the OFC, and the test was repeated
[67]
. This mouse exhibited dominance
in 22 out of 50 trials. A positive control
test was also conducted to compare the
efficacy of BZN. A pair of mice sized
similarly to those in the previous test
Figure 8: Effects of Drug Administration on Shock-Induced Aggression. The method of measuring percentage of attacks/5o shocks was based on previous shockinduced aggression tests [61]. The average percentages
(over three days) were calculated before and after the
administration of the drug to one mouse per pair. BZN
reduced the number of attacks from 22.4% to 11.5% ,
whereas serotonin reduced the number of attacks from
21.1% to 16.0%.
were selected. One mouse dominated
36 out of 50 trials. The dominant mouse
was treated with exogenous serotonin via
cannula (Strategic Applications Inc.) to
the OFC. The serotonin-treated mouse
was dominant 28 out of 50 trials [67]. These
trials illustrate that BZN is more effective
in reducing aggression than solely
increasing serotonin concentrations (Fig.
7).
Test B) Shock-Induced Aggression Test:
In baseline testing, mice were randomly
paired and placed into two groups.
Mice were foot-shocked 50 times daily
for three days. Aggressive attacks and
counterattacks were measured, and the
percentage of daily attacks/50 shocks was
calculated and averaged over the three
days [58, 62]. In one group, one mouse from
each pair was administered BZN, while
in the other group, one mouse per pair
was treated with exogenous serotonin via
cannula (Strategic Applications Inc.) to
the OFC [63]. Mice were tested for shockinduced aggression in a similar manner
to the baseline testing for the three days
following drug administration. The results
indicate that BZN was more successful
in reducing shock-induced aggression
compared to exogenous serotonin
administration (Fig. 8).
FUTURE PLANS AND JYP
Indra Pharmaceuticals has identified
BZN, a groundbreaking agent that
assuredly possesses the capability to
cure wrath. However, it requires further
funding for additional refinement to make
our proposed drug, Chillpil, within which
BZN will be the main active ingredient.
Several tests have been conducted with
BZN to ensure minimal disruption of
peripheral biological pathways. It has been
proven that BZN binds to and positively
allosterically modulates the PS 5-HT1A
receptor with efficacy and specificity.
Furthermore, BZN significantly reduces
aggressive behaviours in vivo.
Chillpil is a drug that has great potential
for commercial use, but it requires
further funding to enhance its specificity
to the OFC, develop methods of viable
administration, and conduct human
clinical trials. Studies have shown that,
through the use of high-throughput
combinatorial
techniques,
certain
agonists can be modified to exhibit brain
region specificity due to selectivity for
unique signal transduction mechanisms
localized in particular areas of the brain
[28,42]
. Additionally, although initial
delivery to rat test subjects has occurred
via cannula, chemical analysis has
determined that BZN can be absorbed into
the bloodstream from the intestines and
cross the blood-brain barrier. This reveals
the favourable likelihood for ChillPil to
be ingested by patients in capsule form.
Finally, ChillPil requires clinical testing
on wrathful populations which would
further corroborate the effectiveness of
this drug.
Violent behaviours are detrimental to
society, and ChillPil will prove to be greatly
beneficial in reducing such instances.
During 2010, Statistics Canada reported
over 437,000 violent incidents across
the country, while the United States
police services reported a grand total of
778,901 aggravated assaults and 14,648
violent homicides [64,65]. Evidently, violent
behaviour is a major public concern, and
greatly impacts both personal and public
safety. Indra Pharmaceuticals foresees
potential markets including psychiatric-
related bail conditions, high-security
prisons, and psychiatric clinics. The
prospect of Chillpil as a pharmaceutical
agent is promising, sure to increase
FUNSIN’s annual profits and greatly
benefit society. The cure for wrath is
tangible, within reach, and presents a
favourable opportunity to build safer
communities.
Part 3.5: Appendix
In brief, below is the general in-silico design protocol for BZN:
1) Identification of suitable 3D-crystal structure to model the 5-HT1A
protein sequence
High-accuracy selection of comparable structures for 5-HT1A was accomplished
through analyzing the evolutionary relationships of the 5-HT1A sequence with those
of all other proteins [30]. Phyre2 system was used to automatically generate and
cross-compare the Hidden Markov Model (HMM) of the 5-HT1A human sequence
with those of all known protein 3D-crystal structures [30,31]. Turkey Beta-1 Adrenergic
Receptor (TBAR1) bound to Cyanopindolol (PDB #2VT4) was shown to be the
highest-scoring structure with 100% confidence in structural prediction and 39%
sequence identity conservation (an identity of 30% or above is considered to be a
highly accurate structural prediction) [30].
2) Identifying alternate conformations of 5-HT1A
I) Identification of other 3-D crystal structural conformers in PDB databank to
TBAR-Cyanopindolol crystal structure. These conformers are the same receptor
bound to a high-affinity agonist, high-affinity antagonist, and high-affinity inverse
agonist. These represent a broad sample of the “stable” equilibrium states of 5-HT1A
[31]
.
II) Performing Molecular Dynamic (MD) Trajectory Analysis to identify unstable
transitional forms of 5-HT1A that lie between stable equilibrium conformation
states The identification of these unstable forms using Amber helped to highlight
potential binding sites not exposed in the stable conformations on either side of the
equilibrium [32-34]. The transient nature of such sites may also point towards possible
allosteric behaviour [36,66].
3) Identifying transient solvent-accessible binding sites
Fragment-based analysis of both the stable conformational ensemble (SDE) and
intermediate MD ensemble (IME) was performed using FTMap [34]. The program
analyzed each structure with fragments of common organic solvents (probes), and
determined regions of both high- and low-probe affinity. Sites that were identified as
having, on average, low solvent accessibilities, but large variances within and across
the ensembles were identified as possible sites of transient nature (a key aspect of
potential allosteric regions) [36, 66].
4) Identifying energetically favourable potential binding sites
AutoDock 4 and Autodock Vina (3D-grid based binding affinity analysis) were
employed against all ensembles to determine the pockets to which 8-OH-DPAT (a
high-affinity 5-HT1A agonist) would favourably bind [38-41,67-69]. Pockets with affinity
values (kcal) and other factors above the adequate threshold were considered
energetically favourable binding sites [36,66,68].
5) Identifying allosteric binding pockets
Potential novel binding sites were selected by cross-referencing binding pockets
that satisfied both energetically favourability and solvent accessibility requirements
across SDE and IME sets [36].
6) Determining functional characteristics of allosteric binding pockets
Selecting pockets that would enhance the endogenous activation of the 5-HT1A
receptor, by manually analyzing the stereochemical and residue interaction
properties of each site between SDE and IME sets in relation to structural changes
in the helices and intra- and extracellular domains and binding regions of 5-HT1A [36,
70]
. This allowed for the identification of possible structural changes seen between
different conformations, which were caused by binding to key residues.
Figure 1: 5-HT1A allosteric sites (sites 1 & 4 are negative, sites 2 & 3 are positive) [36]
Figure 2: Allosteric site #2 highlighting key interacting residue Trp-364 [36]
Figure 3: Allosteric site #3 highlighting key interacting residue Glu-147 [36]
6) Building a hybrid structural model
Desmond was used to incorporate SDE and MDE flexibility into a single structural
model [71-75].
Figure 4: MDE and SDE Hybrid Model [36]
7) Performing high-throughput virtual screening to identify lead
compounds that bind to both allosteric sites
The ZINC Clean Lead-like library was blind-docked via high-throughput virtual
structure-based screening against the hybrid 5-HT1A structural model (specifically
localizing the docking targets as the key residues forming the binding pockets of the
positive allosteric sites), followed by further higher precision runs to successively
limit top-scoring results [42, 76]. The ZINC Clean Lead-like library is a database of
core structures that could make up more complex drug-like compounds [42]. These
leads were ideal in that they can be easily modified and added onto to enhance their
selectivity or functional properties. DOCK Blaster was the program used to virtual
screen.
6) Identifying top-scoring lead compound
FIGURE 5: The lead
commpound 2-(2H-1,4benzoxazin-3-ylamino)
benzonitrile.
The top dock-scoring lead compound that also satisfied
high energetic (using a second Autodock run with the
compound) and structural favourability for binding to both
selected allosteric binding pockets, as well as low energetic
favourability for binding to other regions of 5-HT1A, and in
addition, satisfying Lipinski’s Rule of 5 and having ability to
cross the blood-brain barrier, was chosen to continue as the
primary lead candidate [40, 41,66-68]. With further funding, we
believe this lead molecule can be further modified through
high-throughput combinatorial techniques to enhance its
brain region specificity and also to further increase its efficacy
[42]
.
Part 4: Annotated References
(1) de Almeida RM, Ferrari PF, Parmigiani S, Miczek KA. Escalated aggressive behavior: dopamine, serotonin and GABA. European Journal of Pharmacology. [Online]
2005;526(1-3): 51-64. Available from: doi:10.1016/j.ejphar.2005.10.004 [Accessed
20th January 2012].
This review article summarizes research on aggression in animals and humans. It
focuses on the roles of the dopaminergic, GABAergic and serotonergic systems while
pointing out particularly promising methods of intervention. GABA has been shown
to have an inhibitory role in aggression, as studies involving rats have shown that
low levels of GABA in brain areas such as the striatum and the olfactory bulb make
certain rats more susceptible to chronic aggression. In particular, the GABAA receptor may be a plausible target for intervention. In regards to dopamine, research has
proven that dopamine is positively correlated with wrathful behaviour. As such, the
dopamine D2 receptor antagonist haloperidol has been prescribed for decades to
treat highly aggressive behaviour, especially in those with psychosis, however it also
has sedative and locomotion-impairing effects. Also, individuals with low MAOA
activity due to a mutation in the MAOA gene were shown to be more susceptible to
aggressive behaviours. Finally, serotonin (notably in the prefrontal cortex) has an
important inhibitory role in aggression, with the 5-HT1A and 5-HT1B receptors being
of possible importance for treatment. Most of the brain locations that are abundant
in GABAA receptor subtypes are also abundant in serotonergic neurons. This article
contributed valuable background information in the creation of the introduction.
(2) Bjork JM, Moelle FG, Kramer GL, Kram M, Suris A, Rush AJ, Petty F. Plasma
GABA levels correlate with aggressiveness in relatives of patients with unipolar
depressive disorder. Psychiatry Research. [Online] 2001;101(2): 131-136. Available
from: doi:10.1016/S0165-1781(01)00220-7 [Accessed 20th January 2012].
This article establishes the inverse relationship between GABA levels and aggressiveness based on a study of 77 psychiatrically healthy adults. The aggression was measured using the Burkee Hostility Inventory and tested subjects with a first relative
who had primary unipolar depressive disorder. These subjects displayed the negative
correlation between GABA levels and aggressive behaviour, thus possibly indicating
a genetic factor. The relationship was not observed in subjects without this kind of
family history. This information was useful for identifying possible loci for intervention.
(3) Nelson RJ, Trainor BC. Neural mechanisms of aggression. Nature Reviews
Neuroscience. [Online] 2007;8: 536-546. Available from: doi:10.1038/nrn2174
[Accessed 21st January 2012].
This review article defines aggression as an umbrella term for behaviours that are
intended to inflict harm. It summarizes types of aggression, previous methods of
intervention and the possible neural mechanisms involved. Studies suggest that the
reduced activation of the prefrontal cortex are related to heightened levels of aggression. Molecular approaches to studying aggression have revealed biological pathways and signals that mediate these types of behaviours that are possible targets for
therapeutic intervention. Two examples of neuroleptic drugs are chlopromazine and
haloperidol but they are sedative and have many side effects. Newer drugs include
risperidone, which antagonizes serotonin 5-HT2 and dopamine D2 receptors but it
has many unwanted side effects as well.
The article discussed many different molecules associated with aggression, with the
main ones being serotonin, dopamine, norepinephrine, and GABA. In terms of the
serotonergic pathway, low 5-HT levels were associated with increased impulsivity
and aggression. Activation of 5-HT1B heteroreceptors and 5-HT1A receptors have led to
reduced aggression. Dopamine has also been implicated in aggression, with an excitatory role. However, it has been shown to have different effects during development
and in adults. There is less evidence for the positive relationship of norepinephrine
and aggression, as the alpha- and beta-adrenergic receptors elicit different effects.
Although the postsynaptic beta-receptor antagonist propranolol reduced aggression,
both agonists and antagonists were able to elicit the same response on the alpha-2
receptors. Dopamine beta-hydroxylase knockout mice (who cannot produce norepinephrine) showed reduced aggressive behaviours.
Other molecules discussed include MAOA, nitric oxide and steroid hormones but
these molecules did not have significant roles in comparison to the previously discussed molecules. Furthermore, this article discusses the role of different brain
regions in the regulation of wrath. It also shows how those who show high measures
of reactive aggression have lower-than-average baseline activity in the frontal cortex.
Studies suggest that the role of the prefrontal cortex is to provide inhibitory inputs to
circuits to the amygdala, which promotes aggression. As such, this article provided
valuable information regarding the brain circuitry involved in producing aggressive
behaviour and consequently allowed us to deduce which brain regions we were interested in targeting or looking into during our future research regarding molecular
pathways. Due to the substantial evidence regarding the relationship between malfunctionary activity of the PFC and aggression, we narrowed down our target brain
region to the orbitofrontal cortex.
(4) Sanchez C, Arnt J, Hyttel J, Moltzen EK. The role of serotonergic mechanisms in
inhibition of isolation-induced aggression in male mice. Psychopharmacology. [Online] 1993;110(1-2): 53-59. Available from: doi: 10.1007/BF02246950 [Accessed 25th
January 2012].
This article discusses the role of serotonin in aggression while detailing many of the
drugs already available to treat aggression. 5-HT1A agonists have been able to reduce
aggressive behaviours, while the 5-HT2 agonists is only effective at high doses. Tests
involving many types of adrenoceptor agonists have proven their antiaggressive
effects. However, many drugs were also unsuccessful, including mixed 5-HT1A and
beta-adrenoceptor antagonists and 5-HT2 and 5-HT3 antagonists. In tests with isolated male rats, both D1 and D2 receptor antagonists (SCH 23390 and emonapride,
respectively) were unable to reduce aggression. Overall, more information is needed
about the adrenergic system and dopamine antagonists seem ineffective. Serotonin,
and more specifically the 5-HT1A receptors, seem to play an important role in aggression. This article helped to narrow down a choice for drug intervention.
(5) Soyka M. Neurobiology of aggression and violence in schizophrenia. Schizophrenia Bulletin. [Online] 2011;37(5): 913-920. Available from: doi:10.1093/schbul/
sbr103 [Accessed 24th January 2012].
Catcehol-O-methyltranferase is involved in the metabolism of several catecholamines
including dopamine, a key neurotransmitter involved in both schizophrenia and aggression. Many studies suggest that the Val158Met single nucleotide polymorphism
of this gene affects COMT activity, causing the methionine homozygote schizophrenic patients to show a 4- to 5-fold lower COMT activity than in valine homozygotes.
This leads to a higher risk of aggressive and violent behaviours. Positron emission
tomography and single photon-emission computed tomography data indicate deficits
of COMT in both the orbitofrontal and temporal cortex. This article outline a possible
location for intervention, but was limited in its target population.
(6) Reuter M, Weber B, Fiebach CJ, Elger C, Montag C. The biological basis of anger:
associations with the gene coding for DARPP-32 (PPP1R1B) and with amygdala volume. Behavioural Brain Research. [Online] 2009;202(2): 179-183. Available from:
doi: 10.1016/j.bbr.2009.03.032 [Accessed 21st January 2012].
This article discusses a large clinical study that highlighted a possible connection
between a mutated DARPP-32 gene and amygdala volume, with increased potential
to express anger and develop psychotic conditions such as schizophrenia. A C to T
single nucleotide polymorphism (rs907094) of the DARPP-32 gene was found to be
directly related to these kinds of disorders. Another study that attempted to find a
correlation between the rs907094 polymorphism found that carriers of the T-allele
had significantly higher ANGER scores, which was assessed using the Affective Neuroscience Personality Scale. It also provided information on the proposed relationship between anger and decreased activity of catechol-o-methyl transferase (COMT)
and monoamine oxidase A (MAO-A). This study provided additional evidence for
examining dopamine as a possible locus for intervention.
(7) Eichelman BR. Neurochemical and psychopharmacologic aspects of aggressive
behavior. Annual Review of Medicine. [Online] 1990;41:149-158. Available from:
http://www.annualreviews.org/doi/pdf/10.1146/annurev.me.41.020190.001053
[Accessed 10th February 2012].
This review article summarizes several neurotransmitter pathways involved in aggressive behaviour and served as an excellent source of concise background information. It includes five main pathways: acetylcholine, gamma-aminobutyric acid, dopamine, norepinephrine, and serotonin.
(8) Archer J. Testosterone and human aggression: an evaluation of the challenge hypothesis. Neuroscience and Biobehavioral Reviews. [Online] 2006;30(3): 319-345.
Available from: doi:10.1016/j.neubiorev.2004.12.007 [Accessed 13th February 2012].
This review gathered information from a multitude of studies and saw how the challenge hypothesis applied to humans. Originally, the challenge hypothesis relates
testosterone to evolution and works well with the “mouse model” as well as other
animals. However, it was found that not all the points were valid when applied to humans. In the specific aspect of aggression, it was found that various studies reported
a low-to-no correlation between testosterone levels and aggression. Some specific
cases such as testosterone in prepubescent boys were stronger and in general, the
positive correlation of testosterone and aggression in females was higher. However,
it is clear from the review that much work needs to be done and that there are many
factors that play into testosterone and aggression. Thus, our decision to not target
testosterone is well-supported. The article is very specific with articles and past studies and uses many in order to make a point. Thus, the arguments are well supported
and credible. The review has also been cited over 200 times and has thus, been verified as credible.
(9) Mehta PH, Beer J. Neural mechanisms of the testosterone-aggression relation: the role of orbitofrontal cortex. Journal of Cognitive Neuroscience. [Online]
2010;22(10):2357-2368. Available from: doi:10.1162/jocn.2009.21389 [Accessed
20th February 2012].
This article studies the pathway associated with aggression and testosterone using
a decision-making test where participants had to make a social decision. The study
was a combination of literature showing a correlation between testosterone, the
orbitofrontal cortex and aggression separately. Neuroimaging techniques were used
in combination with salivary samples to measure hormones. A variety of neurons
were activated during aggressive decisions, such as the caudate nucleus and anterior
cingulate cortex but the most frequent was the medial orbitofrontal cortex. Thus, this
study gives support to testosterone as a contributor to aggression and also proposes
a possible neural pathway through the medial orbitofrontal cortex. The journal is associated with the Massachusetts Institute of Technology, a renowned institution and
has an impact factor of 5.357 during that year.
(10) O’Connor DB, Archer J, Wu FC. Effects of testosterone on mood, aggression and
sexual behaviour in young men: a double-blind, placebo-controlled, cross-over study.
The Journal of Clinical Endocrinology & Metabolism. [Online] 2004;89(6): 28372845. Available from: doi: 10.1210/jc.2003-031354 [Accessed 15th February 2012].
This study used clinically appropriate levels of testosterone and injected them over
four weeks and then monitored a variety of behaviours using questionnaires and
assays. Pertaining to aggression, they used questionnaires filled out by the volunteer, their partner and an activity-based test. Their results showed that although
testosterone has been associated with aggression in past studies, those studies used
extremely high doses of testosterone. The changes observed in aggressive behaviour
were mostly insignificant and given that there were twenty eight volunteers, it would
require a greater population to confirm a correlation. Therefore, the article lends
support to our choice in serotonin since testosterone is not confirmed to be related to
aggression. The journal is in association with the Endocrine Society and the method
is very clearly laid out. Other studies are also referenced frequently and properly to
validate their information. They compare and contrast their findings to others. Furthermore, it has been cited by several articles from a wide variety of journals. Thus,
the article is verified.
(11) Montoya ER, Terburg D, Bos PA, Honk JV. Testosterone, cortisol, and serotonin as key regulators of social aggression: a review and theoretical perspective.
Development and Psychopathology. [Online] 2004;16(1): 69-93. Available from:
doi:10.1007/s11031-011-9264-3 [Accessed on 20th February 2012].
This is a highly detailed article published in a reputable journal that provides a
thorough investigation of the possible underlying molecular mechanisms of wrath.
It specifically outlines various studies that attribute low levels of cortisol, high levels
of testosterone, and low levels of serotonin to aggressive behaviour. In rat systems,
lower levels of endogenous cortisol in relation to higher testosterone levels were seen
to attribute to chronic aggression. However, administering cortisol to rats with low
endogenous cortisol increased anger. Thus, the mechanism is more complex than
simply low cortisol and high testosterone. This article supports the decision to avoid
targeting both these molecules since using artificial forms of cortisol and testosterone
trigger a different mechanism from endogenous forms.
(12) Herrero N, Gadea M, Rodriguez-Alarcon G, Espert R, Salvador A. What happens
when we get angry? Hormonal, cardiovascular and asymmetrical brain responses.
Hormones and Behaviour. [Online] 2010;57(3): 276-283. Available from: doi:
10.1016/j.yhbeh.2009.12.008 [Accessed 18th February 2012].
This article attempted to trigger anger in healthy young men in order to observe the
physiological and emotional changes that occur. They connected not only salivary
testosterone and cortisol to anger induction but also the activity of brain structures.
However, the latter is not particularly relevant to our research. The study found that
anger induction caused an increase in self-reported anger, salivary testosterone and
a decrease in cortisol levels. However, the study does not exactly point to a cause and
effect relationship. Rather, it establishes that at least there is a common cause relationship. This article was also evidence as to why we chose not to target the testosterone pathway. The article cites many previous studies and explores all previously
established hypotheses and thus, shows more credibility. The impact factor of the
journal over five years has been high, showing that the journal is reputable.
(13) Terburg D, Morgan B, van Honk J. The testosterone-cortisol ratio: a hormonal
marker for proneness to social aggression. International Journal of Law and Psychiatry. [Online] 2009;32(4): 216-223. Available from: doi: 10.1016/j.ijlp.2009.04.008
[Accessed 19th February 2012].
This review uses the triple balance of emotion model (three levels of effect) to analyze how social aggression is linked to high testosterone and low cortisol ratios. The
review makes a convincing argument for how the model applies to social behaviour.
However, most of the referenced studies were published in the nineties and the model was intended for psychopathy. The review gives support to testosterone and cortisol as a target for our research. However, it conflicts with a variety of other studies
and reviews. Furthermore, both have long term effects that are unrelated to aggression. Also, the review does mention serotonin as a mediator for impulsive aggression.
The review is published in a credible journal and cites other articles frequently and
appropriately. It has been referenced by over 20 other journals.
(14) Coccaro EF. Impulsive aggression and central serotonergic system function in
humans: an example of a dimensional brain-behavior relationship. International
Clinical Pharmacology. [Online] 1992;7(1):3-12. Available from: http://www.ncbi.
nlm.nih.gov/pubmed/1624755 [Accessed 11th March 2012].
This review outlines all the studies that have found a correlation between the role
of the serotonergic system and the regulation of aggression in both animals and
humans. It discuss both behaviour and correlative studies, indicating that there is
evidence of this connection at both the cellular level and in functional clinical testing.
The author, Coccarro, has extensively studied the role of serotonin in the modulation
of violent behaviour. He has conducted and published numerous reviews regarding
the information on this topic thus making it a highly credible and thorough source.
(15) Ferrari PF, Palanza P, Parmigiani S, de Almeida RMM, Miczek KA. Serotonin
and aggressive behavior in rodents and nonhuman primates: predispositions and
plasticity. European Journal of Pharmacology. [Online] 2005;526(1-3):259-273.
Available from: doi: 10.1016/j.ejphar.2005.10.002 [Accessed 12th March 2012].
This review was used as evidence that the link between serotonin and aggression has
been found in rodents and non-human primates. Although this article is mainly look-
ing into genetic predispositions to wrath, it does focus on the role of serotonin in this
evolutionary pathway. Importantly, it outlines and mentions multiple articles supporting the link between a serotonin deficiency and increased aggressive behaviour,
through the measurement of the serotonin metabolite, CSF 5-HIAA. Of these articles,
the studies vary from rats to monkeys to nonhuman primates, supporting the claim
that this correlation is widespread.
(16) Coccaro EF, Siever LJ, Klar HM, Maurer G, Cochrane K, Cooper TB, et al. Serotonergic studies in patients with affective and personality disorders. Correlates
with suicidal and impulsive aggressive behavior. Archives of General Psychiatry.
[Online] 1989;46(7):587-599. Available from: http://www.ncbi.nlm.nih.gov/pubmed/2735812 [Accessed 25th March 2012].
This study was used in conjunction with “Serotonin and aggressive behaviour in
rodents and nonhuman primates”. Both papers evidence that decreased serotonergic
function is correlated with increased aggressive behaviour, as indicated by cerebral
spinal fluid levels of the serotonin metabolite. This article however is a study, not a
review, and it was conducted on humans. This helps to justify the serotonergic pathway as a valid loci for intervention, as rodent and non-human results do not always
transfer to clinical trials. The link observed in this study was found in a variety of humans including psychopaths, violent offenders, suicidal patients and nonpsychiatric
individuals, indicating that with this locus comes a variety of wrathful patients.
(17) Sala M, Caverzasi E, Lazzaretti M, Morandotti N, DeVidovich G, Marraffini E, et
al. Dorsolateral prefrontal cortex and hippocampus sustain impulsivity and aggressiveness in borderline personality disorder. Journal of Affective Disorders. [Online]
2011;131(1-3):417-421. Available from: doi:10.1016/j.jad.2010.11.036 [Accessed 23rd
March 2012].
This journal article described how patients with borderline personality disorder have
increased levels of aggression, and are unable to control impulsive behaviour due to
varying volumes of the hippocampal region and the dorsolateral prefrontal cortex.
This article was used as evidence to depict that there are many regions within the
prefrontal cortex that regulate human aggression.
(18) Blair RJR. The roles of orbital frontal cortex in the modulation of antisocial
behavior. Brain Cognition. [Online] 2004 6;55(1):198-208. Available from: doi:
10.1016/S0278-2626(03)00276 [Accessed 28th March 2012].
This study reveals that the orbital frontal cortex modulates the subcortical region,
which has been proven to be directly related to the regulation of aggressive behaviour. While the orbital frontal cortex does not inhibit aggression, damage to this
region has been suggested to be involved in socially inappropriate and aggressive
behaviour. Sensory information in the form of ‘anger stimuli’ is received at the orbital frontal cortex, and then travels to the striatal nucleus of the amygdala and to
the medial hypothalamus. Subsequently, the information is translated to the dorsal
periaqueductal gray and observed at the behavioural level as violence and aggression.
This article helped to validate the role of the orbital frontal cortex in the modulation
of aggressive behaviour.
(19) Puig MV, Gulledge AT. Serotonin and prefrontal cortex function: neurons, networks, and circuits. Molecular Neurobiology. [Online] 2011; 44(3): 449-464. Available from: doi: 10.1007/s12035-011-8214-0 [Accessed 25th March 2012].
This article provides evidence of the abundance of 5-HT1A, 5-HT2A and 5-HT3A receptors in the prefrontal cortex (PFC). In fact, it states that 60% of rat PFC neurons
express 5-HT1A and 5-HT2A receptors. Most of these neurons (up to 80%) coexpress
these receptors, meaning that the 5-HT1A and 5-HT2A receptors function co-actively
in order to mediate neurochemical responses. Since 5-HT1A receptors are inhibitory, whereas 5-HT2A receptors are excitatory, it was studied further how exactly the
5-HT1A receptor activity impacts 5-HT2A activity (or vice versa). Results showed that
majority of adult pyramidal neurons functionally are inhibited by 5-HT in a 5-HT1A
dependant manner, implying that 5-HT1A activity serves a more dominant role in
controlling neurotransmission whereas 5-HT2A receptors allow for fine-tune adjustments of these responses. Furthermore, 5-HT3A receptors have been found on superficial layers of the prefrontal cortex, and provides evidence in regards to how this
layer is not as involved in emotion-control functions of the PFC [33]. The thorough
investigation of each of these receptors’ roles in the PFC provided a method to narrow down our focus to one receptor - the 5-HT1A postsynaptic receptor.
(20) Cleare AJ, Bond AJ. Ipsapirone challenge in aggressive men shows an inverse
correlation between 5-HT1A receptor function and aggression. Psychopharmacology. [Online] 2000;148(4):344-349. Available from: doi: 10.1007/s002130050061
[Accessed 12th February 2012].
This study aimed to validate the suggestion that 5-HT1A receptor function is linked
to aggression, and found positive results. The study involved 12 healthy men who
were selected to have high levels of wrathful behaviour. They were administered
ipsapirone which is a 5-HT1A receptor partial agonist, and it was observed that subjects with impaired neuroendocrine response to the agonist had high self ratings of
aggression. This article was used as justification for choosing to modulate the 5-HT1A
receptor, as it provided evidence that increasing the function of the 5-HT1A receptor
would have a positive effect on reducing aggression. It also mentions that this correlation is seen both within the health range and the personality-disorder range. This
encouraged us to pursue this pathway as our drug could be marketable to a broad
population.
(21) Goodfellow NM, Benekareddy M, Valdya VA, Lambe EK. Layer II/III of the
prefrontal cortex: inhibition by the serotonin 5-HT1A receptor in development and
stress. The Journal of Neuroscience. [Online] 2009;29(32): 10094-103. Available
from: doi:10.1523/​JNEUROSCI.1960-09.2009 [Accessed 25th March 2012].
This article discusses how decreased 5-HT1A activity (in layers II/III of the PFC) during stages of development have been shown to be associated with mood and psychiatric disorders such as schizophrenia. This provides evidence about the role of 5-HT1A
receptors in deeper layers of the PFC in emotive controls. In particular, serotonin
in these areas of the brain region functions to inhibit pyramidal neurons through
5-HT1A receptor binding. This agrees with reference 19, which states that 5-HT1A inhibits neurotransmission, confirming that 5-HT1A activity plays a dominant function
in the inhibition of neurons resulting in increased emotional control. There was also
a thorough explanation in this article about the experimental procedure on prefrontal cortex slices of rats that produced such results. Finally, this article also confirmed
that the 5-HT1A response in the PFC is mediated through activation of GIRK channels, supporting the background information in article reference 43 that explained
the activation of GIRK through g-protein coupled activation by the 5-HT1A receptors.
Overall, this article provides sufficient confirmatory evidence that allowed for further
justification for the choice of 5-HT1A receptors due to their prominent role in PFCmediated control over emotions such as anger.
(22) Vry JD. 5-HT1A receptor agonists: recent developments and controversial issues. Psychopharmacology. [Online] 1995;121(1):1-26. Available from: doi: 10.1007/
BF02245588 [Accessed 14 February 2012].
This article outlines the effects of agonizing presynaptic autoreceptors vs. postsynaptic heteroreceptors in various region of the brain. Through testing agonists on different regions, behavioural results exhibited that postsynaptic 5-HT1A was most involved
in antidepressive effects (whereas presynaptic 5-HT1A is more associated with anxiolytic effects). Furthermore, it also discusses how presynaptic 5-HT1A receptors control
general 5-HT neuronal activity. Recent studies indicate how the presynaptic 5-HT1A
receptors in the cortex are involved in a negative feedback loop, in which increasing
agonist activation of these receptors results in lower 5-HT synaptic concentration.
This supports the idea stated in the report that outlines the negative effects that can
result from targeting these presynaptic receptors.
(23) Schloss P, Williams D. The serotonin transporter: a primary target for antidepressant drugs. Journal of Pyschopharmacology. [Online] 1998;12(2):115-121. Available from: jop.sagepub.com [Accessed 27th February 2012].
This source was used for background information on the major classes of antidepressant drugs. It is a review article that classifies modern drugs and discusses the mechanisms of the most widely used antidepressants. It was effective in discerning which
classes of drugs are being prescribed currently, as well as historically. This article was
published in a scientific journal on behalf of the British Association for Psychopharmacology. An extensive, properly cited reference list was also provided, adding to its
credibility.
(24)Fuller RW, Wong DT. Effects on antidepressants on uptake and receptor systems
in the brain. Progress in Neuro-Pyschopharmacology and Biological Psychiatry.
[Online] 1985;9(5-6):485-490. Available from: doi: 10.1016/0278-5846(85)9006-5
[Accessed 27th February 2012].
Fuller and Wong describe the most common SSRIs and their effects on neuronal systems when prescribed for depression. This site was useful in determining the longterm effects of prolonged use of SSRI medication while outline its negative aspects. It
gives much detail on the adverse effects of another class of antidepressants: tricyclic
antidepressant drugs and. This is likely due to the funding by Lilly Research, a pharmaceutical company which produces the most widely prescribed SSRIs.
(25) Heller JL, Zieve D. Serotonin Syndrome. [Online]. Available from: http://www.
ncbi.nlm.nih.gov/pubmedhealth/PMH0004531/ [Accessed 17th February 2012].
This encyclopedia page from PubMed Health provides information about serotonin
syndrome, such as its causes and its symptoms. Serotonin is a potentially fatal response due to excessive serotonin levels in the body. This usually occurs when certain
drugs are taken in conjunction with selective serotonin reuptake inhibitors (SSRIs)
which increase the concentration of synaptic serotonin. Various symptoms of serotonin syndrome include restlessness, fever, increased heartbeat and rapid blood pressure. This information is provided by the National Center for Biotechnology Information, a renowned source for peer-reviewed scholarly journal articles. This supported
our use of postsynaptic intervention, since autoreceptor intervention can lead to
serotonin syndrome.
(26) Baud P, Perroud N, Courtet P, Jaussent I, Relecom C, Jollant F, Malafosse
A. Modulation of anger control in suicide attempters by TPH-1. Genes, Brain
and Behavior. [Online] 2009;8(1): 97-100. Available from: 10.1111/j.1601183X.2008.00451.x [Accessed on 20th February 2012].
This study investigated the association between the A218C polymorphism of the
tryptophan hydroxylase gene and suicidal behaviour. It was found that suicide attempters carrying the AA genotype scored significantly lower on the Anger Control
subscale than those with AC or CC genotypes, indicating the effect of this polymorphism on violent behaviours. This article was used as an example of gene mutations
affecting the serotonergic pathway.
(27) Khan, A. Vilazodone, a novel dual-acting serotonergic antidepressant for
managing major depression. Expert Opinion of Investigational Drugs. [Online]
2009;18(11): 1753-64. Available from: doi:10.1517/13543780903286396 [Accessed
12th February 2012].
Vilazidone is a dual-acting drug that is both a 5-HT1A partial agonist and a SSRI with
useful applications in the context of treating depression. In addition to activating
5-HT1A, vilazidone increases synaptic levels of 5-HT in areas such as the hippocampus and cortex, and there is no way of selectively administering this drug to target
the specific areas of the brain or to specific receptors associated with anger (prefrontal cortex, hypothalamus, amygdala). The low efficacy of this drug in activating
the 5-HT1A receptor also averts the possibility of serotonin syndrome, but may not
produce significant enough results in the context of wrath. Thus we did not wish to
look further into the development of a combination of a SSRI/5-HT1A partial agonist
similar to vilazidone due to the the larger scale effects on serotonin and the low efficacy of the drug.
(28) Depoortere R, Auclair AL, Bardin L, Colpaert FC, Vacher B, Newman-Tancredi
A. F15599, a preferential post-synaptic 5-HT1A receptor agonist: activity in models of cognition in comparison with reference 5-HT1A receptor agonists. European
Neuropsychopharmacology. [Online] 2010;20(9): 641-54. Available from: doi:
10.1016/j.euroneuro.2010.04.005 [Accessed 13th February 2012].
F15599 is a molecule with a high affinity specifically for post-synaptic 5-HT1A receptors located in select regions of the brain, such as the prefrontal cortex (PFC). It is
also a high efficacy 5-HT1A agonist that specifically activates 5-HT1A post-synaptic
receptors, yet requires a 10-fold increase in dosage in order induce the toxic effects of
serotonin syndrome. It is proposed that the selectivity of this ligand to only 5-HT1A
post-synaptic receptors in the PFC is due to preferential activation of 5-HT1A receptors that evoke GIRK-related responses (see reference 44 for different kinds of serotonin signal trandsuction response pathways). Furthermore, the F15599 molecule
binds to 5-HT1A receptors that possess the g-αi protein subunits (vs. g-αo g-protein
sub units). 5-HT1A receptors in the cortex are more often coupled to gai sub units,
and GIRK-related responses are also associated with selective regions of the brain.
F15599’s two mechanisms of producing brain-region specificity is what we proposed
to utilize in our future steps with our own ligand. The reason that we did not develop
a ligand analogous to F15599 is due to the fact that anger is not necessarily always
caused due to low serotonin levels in aggressive people. Although providing a high-
affinity ligand such as F15599 would definitely produce noticeable wrath-reducing
effects in those who are deficient in 5-HT, it would not be able to address a larger
wrathful population whose symptoms may be a product of 5-HT1A receptor desensitization, or low agonist binding affinity to 5-HT1A receptors.
(29) Barrando S, Sallés J. Allosteric modulation of 5-HT1A receptors by zinc: binding studies. Neuropharmacology. [Online] 2009;56(2): 455-62. Available from: doi:
10.1016/j.neuropharm.2008.09.018 [Accessed 12th February 2012].
Zinc is an allosteric modulator of 5-HT1A receptors that reduce the affinity of the
5-HT1A receptor for both agonists and antagonists. It is suspected to be an interrupting factor in therapy that aims to target 5-HT1A receptor activity. Unfortunately, zinc
is abundantly found in all parts of the brain and especially in the binding sites of
many receptor proteins, and its physiological role in inhibiting the 5-HT1A receptors
itself is not completely understood. Thus, depleting endogenous zinc concentration
was not a liable route of increasing 5-HT1A receptor activity. The example of zinc,
however, illustrated that allosteric modulation of 5-HT1A receptors was a promising
route of action for treating wrath. This article illustrates that molecules like zinc can
decrease receptor-agonist affinity as a result of negative allosteric modulation, thus
naturally decreasing 5-HT1A receptor activity. Development of positive allosteric
modulators that have a greater affinity for 5-HT1A receptors than negative allosteric
modulators such as zinc will allow for a method to increase 5-HT1A activity.
Note: For the following program references, the citation groupings under each program title are required to cite usage of the program.
Phyre2:
(30) Kelley LA, Sternberg MJE. Protein structure prediction on the web: a case study
using the Phyre server. Nature Protocols. [Online] 2009;4:363-371. Available from:
doi:10.1038/nprot.2009.2 [Accessed 23rd February 2012].
Phyre2 is a web server-based program that generates high accuracy structural predictions for inputted protein sequence. It does this by calculating the evolutionary
Hidden-Markov model of inputted proteins and automatically comparing this those
of all other known proteins in the PDB databank. It also ranks by sequence similarity, and optionally ab initio modelling and Poing analysis. This program was critical in
the development of our lead candidate as the crystal structure for 5-HT1A (like many
other GPCRs) has not been determined to date.
Worldwide Protein Data Bank
(31) Berman H, Henrick K, Nakamura H. Announcing the worldwide protein
data bank. Nature Structural Biology. [Online] 2003;10:980. Available from:
doi:10.1038/nsb1203-980 [Accessed 10th March 2012].
The Worldwide Protein Data Bank, and specifically the RCSB subset, provided the
basis for the stable equilibrium protein structures (and the subsequent MD-trajectory models) used in the protocol. It also had other domain analysis functions for
sequence-region comparison that was critical in determining positions of allosteric
sites within the protein (and when the protein is embedded in the cell membrane).
Amber Tools/Amber Lite:
(32) Case DA, Darden TA, Cheatham TE, Simmerling CL, Wang J, Duke RE, et al.
PA. AMBER 11. University of California. 2010
(33) Case DA, Cheatham TE, Darden T, Gohlke H, Luo R, Merz KM, et al. The Amber
biomolecular simulation programs. Journal of Computational Chemistry. [Online]
2005;26:1668-1688. Available from: doi:10.1002/jcc.20290 [Accessed 27th February
2012] .
(34) Ponder JW, Case DA. Force fields for protein simulations. Advances in Protein
Chemistry. [Online] 2006;66:27-85. Available from: http://www.ncbi.nlm.nih.gov/
pubmed/14631816 [Accessed 1st March 2012].
(35) Cheatham TE, Young MA. Molecular dynamics simulation of nucleic acids: successes, limitations and promise. Biopolymers. [Online] 2001;56(4):232-256 Available from: doi:10.1002/1097-0282(2000)56:4<232::AID-BIP10037>3.0.CO;2-H
[Accessed 2nd March 2012].
The Amber suite was used to perform simplified molecular dynamic trajectory analysis. Although our analysis was reduced in comparison to actual studies, such as on
the Beta1 adrenergic receptor, it still provided us with a small ensemble to take into
account the transient nature of the unstable equilibrium states of 5HT1A.
(36) Ivetac A, McCammon JA. Mapping the druggable allosteric space of G-protein
coupled receptors: a fragment-based molecular dynamics approach. Chemical Biology & Drug Design. [Online] 2010;76(3):201-217. Available from: doi:10.1111/j.17470285.2010.01012.x [Accessed 12th March 2012].
This article was an excellent source of information on the potential approaches to
discovering novel allosteric binding sites on G-protein coupled receptors. In particular, the study outlines the use of MD trajectory analysis, along with fragment-based
organic solvent mapping and energetic analysis. Based on the expert knowledge of
the author’s, it also postulated which of the suspected allosteric sites were positive in
nature, and exactly how they exhibited their effect. Overall, it helped to supplement
and confirm elements of the proposed pre-existing protocol developed by our group.
(37) Brenke R, Kozakov D, Chuang GY, Beglov D, Hall D, Landon MR, et al. Fragment-based identification of druggable ‘hot spots’ of proteins using Fourier domain
correlation techniques. Bioinformatics. [Online] 2009;26(5):621-627. Available
from: doi:10.1093/bioinformatics/btp036 [Accessed 26th February 2012].
FTMap is a web-based server program that performed fragment-based accessibility analysis of a protein to a wide variety of organic solvents. In our protocol, it was
used to detect variances in the transient accessibility of various sites across the stable
equilibrium and MD ensembles, through analysis of the fragment probe affinity values.
(38) Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. Journal of Computational Chemistry. [Online] 2009;31(2):455-461 Available from:
doi:10.1002/jcc.21334 [Accessed 13th March 2012].
(39) Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, et al.
AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. Journal of Computational Chemistry. [Online] 2009;31(16):2785-2791. Available
from: doi:10.1002/jcc.21256 [Accessed 12th March 2012].
Autodock 4 and Autodock Vina are programs that determine via the generation of
3D-structural grids, the energetic binding affinity of ligands to an uploaded protein
structure. The program runs off a web-based server cluster. In our protocol, it allowed for the determination of energetically-favourable binding sites on 5HT1A that
could be cross-referenced against dynamic organic solvent fragment accessible sites
to determine overall likely allosteric pockets.
(40) Friesner RA, Murphy RB, Repasky MP, Frye LL,Greenwood JR, Halgren TA, et
al. Extra precision Glide: docking and scoring incorporating a model of hydrophobic
enclosure for protein-ligand complexes. Journal of Medicinal Chemistry. [Online]
2006;49:6177–6196. Available from: doi:10.1021/jm051256o [Accessed 16th March
2012].
(41) Park MS, Gao C, Stern HA. Estimating binding affinities by docking/scoring
methods using variable protonation states. Proteins: Structure Function, and Bioinformatics. 2011;79(1):304-314. Available from: doi:10.1002/prot.22883 [Accessed
3rd March 2012].
The Glide component of the Schrodinger Design Suite is a high-throughput, blinddock virtual screening software. Due to limitations in computing power available, it
could not be used for its original intended purpose in the protocol (which was instead accomplished by DockBlaster). However, it was used to generate preliminary
3D grids and other preconditions that helped us to better understand the structural
nature of the binding pockets as well as various preconditions that should be checked
for binding analyses.
Zinc Database:
(42) Irwin JJ, Shoichet BK. ZINC - a free database of commercially available compounds for virtual screening. Journal of Chemical Information and Modeling. [Online] 2005;45(1):177-182. Available from: doi:10.1021/ci049714+ [Accessed 10th
March 2012].
The ZINC database, specifically the “clean lead-like” subset, was used as the virtualscreening library in our protocol. The clean, lead-like subset provided patented and
unpatented potential lead candidates (along with their basic properties) that could be
easily developed into larger, drug-like compounds. The “clean” designation indicates
that certain compounds not easily experimentally assayed for binding in past studies
were removed.
(43) Polter AM, Li X. 5-HT1A receptor-regulated signal transduction pathways
in brain. Cellular Signalling. [Online] 2010;22(10):1406-1412. Available from:
doi:10.1016/j.cellsig.2010.03.019 [Accessed 20th March 2012].
This article discusses the various signal transduction pathways that occur upon postsynaptic 5-HT1A activation on neurons. It also addresses how certain signal transduction pathways show brain region selectivity. The 5-HT1A pathway involves decreased
cAMP production and decreased PKA phosphorylation and PKA dissociation. This
certain pathway provides information in regards to methods of measuring 5-HT1A
activity addressed in objective 1. Another pathway involves the activation of GIRK
potassium channels in the hippocampus and in the dorsal raphe nucleus, and thus
was not an appropriate method of measuring signal transduction for orbitofrontal
cortex purposes. Other protein-kinase related signalling pathways that were activat-
ed by the 5-HT1A receptors involved mitogen-activated protein kinases (specifically
extracellular signal-regulated kinases, or ERKs) and Akt, both of which were involved
in cell growth and survival. ERK. These two pathways were not further addressed
because studies of the frontal cortex, 5-HT1A receptors have shown to have no effect on phosphorylation of ERK proteins. Furthermore, Akt-activation brain region
specificity has not been studied as thoroughly. The information about the signalling
pathways involved in 5-HT1A post-synaptic activation was summarized in Figure 3 of
the Background Information in the UNsin report.
(44) Nikolaev VO, Lohse MJ. Monitoring of cAMP synthesis and degradation in living cells. Physiology. [Online] 2006;21(2):86-92. Available from: doi:10.1152/physiol.00057.2005 [Accessed 22nd March 2012].
This article discusses the various methods of measuring cyclic AMP in living cells. An
overview is given of the role of cAMP in various capacities such as immune function
and insulin secretion. It also explains methods of measuring cAMP that destroy the
cell. However, since it would be preferable for the neurons to be tested over time, the
methodology needs to monitor living cells. The technique of FlCRhR is introduced
since cAMP causes PKA to dissociate. This is a specific technique modified from
FRET. The regulatory and catalytic subunits are each labelled with a fluorophore and
FRET analysis is used to observe fluorescence. When the subunits are binding, the
individual wavelengths are not observed. However, if cAMP levels increase, the subunits will dissociate and the ratio of the individual wavelengths will increase. Thus,
experiment 1 in objective 1 shows a lesser increase in the ratio of the two emissions
when BZN is introduced since cAMP levels are decreased. The ratio cannot decrease
in the short timespan of the trial since the subunits require multiple steps to reassociate.
(45) Vincent P, Brusciano D. Cyclic AMP imaging in neurones in brain slice preparations. Journal of Neuroscience Methods. [Online] 2001;108(2):189-198. Available
from: doi:10.1016/S0165-027(01)00393-4 [Accessed 25th March 2012].
This study uses the method of FlCRhR to measure the cAMP levels in brain slices.
Neurons have particularly sensitive membranes so the original technique of fluorophore injection would not have been appropriate. However, the group used patch
pipette perfusion to introduce the FlCRhR solution. They also tested cAMP response
to activation using G-protein coupled receptors. In particular, they used a beta-1 adrenergic receptor and then stimulated it. The ratio was found to increase when cAMP
was stimulated. Figure 3C in this study was used as a basis for Figure 4 in objective 1.
It was also discussed that the recovery of the ratio was not immediate after removal
of an agonist. Therefore, for the study, neurons would be pretreated with the adenylyl
cyclase activator but BZN would inhibit the activation of adenylyl cyclase.
(46) Goaillard JM, Vincent P. Serotonin suppresses the slow afterhyperpolarization
in rat intralaminar and midline thalamic neurones by activating 5-HT7 receptors.
Journal of Physiology. [Online] 2002;541(2):453-465. Available from: doi:10.1113/
jphysiol.2001.013896 [Accessed 18th March 2012].
The study used rat thalamic brain neurons in an attempt to discover the pathway for
5-HT7 receptors. When the neurons were flooded with serotonin, the amount of intracellular cyclic AMP was increased in the cytosol. This was measured using the FlCRhR method. The exact methods for preparing the PKA subunits and fluorophores
are highlighted such as optical equipment and concentrations of FlCRhR solution.
Figure 7C graph shows the result of serotonin exposure and thus, provided a basis
for the graph in the results of Figure 4. However, as 5-HT7 receptors are positively
coupled to adenylyl cyclase while 5-HT1A is negatively coupled, the results had to be
altered. Still, the increments of time and fluorescence ratio were crucial in constructing the test graph.
(47) Molecular Probes. Cyclic AMP Fluorosensor (FlCRhR). [Online]. Available from:
http://probes.invitrogen.com/media/pis/mp06660.pdf [Accessed 15th March 2012].
Molecular Probes’ information sheet for their product FlCRhR was used to determine
the function of the probe as well the experimental applications. This showed that the
FlCRhR solution could be purchased commercially for testing and confirmed that its
application was valid in testing BZN’s effects.
(48) EMD Millipore. GPCR Cell Lines. [Online]. Available from:
http://www.millipore.com/search.do?q=GPCR+Cell+Lines#0:0. [Accessed 14th
March 2012].
EMD Millipore, also known as Merck Millipore, was the source of the cell lines purchased for the experiments involving functional readouts of the 5-HT1A and other
receptor. It is a global pharmaceutical and chemical company with a wide range of
products and services in bioscience, lab solutions, and process solutions.
(49) Schmitt JM, Stork PJS. PKA phosphorylation of Src mediates cAMP’s inhibition of cell growth via Rap1. Molecular Cell. [Online] 2002;9:85-94. Available from:
doi:10.1016/S1097-2765(01)00432-4 [Accessed 28th March 2012].
This study sought to regulate cell growth by inhibiting and exciting the cAMP path-
way. It was found in previous research that cAMP activated PKA which activated
tyrosine kinase Src which ultimately antagonized growth factor activation. A specific
downstream protein in the pathway appears to be Rap1 and so the study focused
on how cAMP activated Rap1. To trigger cAMP levels, an adenylyl cyclase activator,
forskolin was used. To determine whether PKA was necessary in the activation of
Rap1, increasing doses of H89 were added while measuring ability to activate Rap1.
Important to objective 1 is the use of H89 as a selective PKA inhibitor. In objective 1,
H89 is used as a positive control since it mimics the reduction of PKA activation. It
has a different mechanism than 5-HT1A receptors but the FlCRhR results should yield
a similar trend. Thus, this article was useful in determining the positive control and
using forskolin to pre-stimulate adenylyl cyclases.
(50) Traish AM, Moreland RB, Gallant C, Huang YH, Goldstein I. G-protein coupled
receptor agonists augment adenylyl cyclase activity induced by forskolin in human
corpus cavernosum smooth muscle cells. Receptors & Signal Transduction. [Online]
1997;7(2):121-132. Available from Pubmed: http://www.ncbi.nlm.nih.gov/pubmed/9392440 [Accessed 28th March 2012].
This study triggered various G-protein coupled receptors that were known to augment adenylyl cyclase while simultaneously using forskolin. Results showed that
the two activation pathways were intertwined and thus, acted on the same adenylyl
cyclases. Objective 1 and 2 use 5-HT1A which is a G-protein coupled receptor and
forskolin simultaneously. However, since 5-HT1A is negatively coupled with adenylyl
cyclase, the effect observed in the study would be opposite to those in the experimental test. Thus, this study supports the use of forskolin to activate adenylyl cyclase that
are also inhibited by 5-HT1A.
(51) Saijo T, Takano A, Suhara T, Arakawa R, Okumura M, Ichimiya T, et al. Effect
of electroconvulsive therapy on 5-HT 1A receptor binding in patients with depression: a PET study with [11C]WAY 100635. International Journal of Neuropsychopharmacology. [Online] 2010;13(6):785-791. Available from: doi:10.3017/
S1461145709991209 [Accessed 23rd March 2012].
This study used WAY 100635 as a 5-HT1A receptor antagonist in a study that measured serotoninergic responses after electroconvulsive therapy. It was found that
ECT did not affect the binding of WAY 100635 and thus, the therapy does not affect
serotonin responses in order to create its antidepressive effect. More importantly, the
study confirms the use of WAY 100635 as an effective 5HT1A postsynaptic antagonist.
This is why it was used in objective 1 as a way to show that an inhibited neuron will
have a greater cAMP concentration and thus the ratio of fluorescence between the
fluorophores should increase.
(52) De Arcangelis V, Liu S, Zhang D, Soto D, Xiang YK. Equilibrium between adenylyl cyclase and phosphodiesterase patterns adrenergic agonist dose-dependent spatiotemporal cAMP/protein kinase A activities in cardiomyocytes. Molecular Pharmacology. [Online] 2010;78(3):340-349. Available from: doi:10.1124/mol.110.064444
[Accessed 25th March 2012].
This study used beta-adrenergic receptors to activate cAMP and protein kinase A
(PKA). FRET was used to measure the response the concentrations of cAMP and
protein kinase in response to isoproternol. Changes in cAMP FRET was positive and
thus, Figure 1C in the study was used as the basis for Figure 5 in objective 2. In that
portion, the rat neurons were treated with various concentrations of BZN and equal
serotonin after adenylyl cyclase activation. FlCRhR was used to measure the ratio of
fluorescence between PKA subunits. As the concentrations of BZN are increased, the
ratio decreased. Thus, this source provided evidence that cAMP and PKA can be dose
dependent and gave a basis for the results graph Figure 5.
(53) Molecular Devices. FLIPR Calcium & Calcium 3, 4, & 5 Assay Kits. [Online].
Available from: http://www.moleculardevices.com/Products/Assay-Kits/GPCRs/
FLIPR-Calcium.html [Accessed 14th March 2012].
This company was the source of the FLIPR Calcium Assay Kits used in testing the
effect of BZN on several GPCRs. The website contained information on how and in
what situations the assay kit would be applicable as well as insight into the mechanism behind the technique. Molecular Devices is an international provider of bioanalytic measurement devices and systems.
(54) Douhan J 3rd, Miyashiro JS, Zhou X, Cole DC, Wu PW, Collins M, et al. A
FLIPR-based assay to assess potency and selectivity of inhibitors of the TEC family kinases Btk and Itk. Assay and Drug Development Technologies. [Online]
2007;5(6):825-838. Available from: doi:10.1089/adt.2007.9982 [Accessed 14th
March 2012]. This article outlines how FLIPR-Based Assays are a valid method of testing the selectivity of a new molecule. In this case, small molecule antagonists were administered
and the assay’s purpose was to test their ability to inhibit tyrosine kinases. Tests were
conducted on cell lines expressing the appropriate kinases. A similar methodology
was adopted in experiment 2 of our testing, as we purchased the appropriate cell
lines and administered BZN to test its ability to create a response. Thus the article
served as evidence that a FLIPR-based assay would be an appropriate test to determine the selectivity of BZN.
(55) Smart D, Jerman JC, Gunthorpe MJ, Brough SJ, Ranson J, Cairns W, et al.
Characterisation using FLIPR of human vanilloid VR1 receptor pharmacology. European Journal of Pharmacology. [Online] 2001;417(1-2):51-58. Available from:
doi:10.1016/S0014-2999(01)00901-3 [Accessed 14th March 2012].
This study conducted a similar FLIPR-based assay to the one in our experimental
methods. We adopted the preparation method and as such, the samples were prepared in 96-well plates, and incubated with the Ca2+ indicator, Fluo-3 (4uM) at
258C for 120min. The goal of this FLIPR assay in their studywas to rank the potency
of various agonists, similar to how we were ranking the potency of BZN on other
receptors. In our case, the results were similar to the -Ca2+ curve in figure 6 of this
study, for all receptor tests, as no response should be seen.
(56) Christiansen B, Meinild AK, Jensen AA, Brauner-Osborne H. Cloning and characterization of a functional human γ-aminobutyric acid (GABA) transporter, human
GAT-2. The Journal of Biological Chemistry. [Online] 2007;282: 19331-19341. Available from: doi: 10.1074/jbc.M702111200 [Accessed 20th March 2012].
Figure 7 of this source was used as a model to create the diagram for experiment 2.
The study conducted a similar FLIPR assay with the aim of testing hGAT-2 against
various receptors and their agonists. Similarly, our diagram showed the effects of
BZN when administered to various receptors and their respective agonists. We also
adopted their method of constructing a dose-response curve based on the maximal
responses observed at various concentrations. In addition, they conducted multiple
tests over three days and averaged these results to one curve. Likewise we created
one average curve for the various receptor subtypes within one neurotransmitter
pathway.
(57) Guo G. Recessive genetic screen for mismatch repair components in BLM-deficient ES cells. PhD thesis. The Wellcome Trust Sanger Institute University of Cambridge. 2004.
This source provides evidence that mice have a similar anatomy and physiology to
that of humans, thus allowing them to act as accurate animal models in biomedical
research, such as drug testing. This study indicates that mice are prevalently used
as models for studying mammalian development, immunology and behaviour. This
source was provided by the Wellcome Trust Sanger Institute in the UK, a leader of
the Human Genome Project. They aim to understand the role of genetics in health
and disease.
(58) Kovacsics CE, Gould TD. Shock-induced aggression in mice is modified by lithium. Pharmacology Biochemistry and Behaviour. [Online] 2010;
4(94):380-386. Available from http://www.sciencedirect.com/science/article/pii/
S0091305709002895 [Accessed 20th February 2012].
This article was used as a source of supplementary information outlining the experimental methodology of conducted a social dominance tube test. In this study,
a single housed-mouse and a group-house mouse entered from the opposite start
sites of the dominance tube. The dominant mouse was indicated as the one forcing
the subordinate mouse back to its start-site. Our testing methods were based upon
this previous test. In addition, this article outlined the condition of the mice prior to
experimentation. This is important in eliminating as much varying external stimuli
as possible, which may contribute to vast differences in baseline aggression between
mice. By conditioning the mice in the same environment, the likelihood of baseline aggression differences due to 5-HT1A concentration or function between mice is
higher. In addition, this study’s experimental method for shock-induced aggression
tests was used. A pair of mice were placed in a Colbourn Habitest Chamber, which is
a clear box with a metal floor through which electrical shocks to the mice are administered. 5 shocks/second at 0.26 mA intensity were delivered to the mice after 2
minutes of social interactions. This study also indicated that mice have been used as
test subjects inshock-induced aggression tests. Aggressive behaviour was recorded
based on the number of direct movements made that resulted in contact, and the
subsequent responses by the attacked rat (ie. biting, sparring).
(59) Stanford School of Medicine Behavioural and Functional Neuroscience Laboratory. Tube dominance test. Available at: http://sbfnl.stanford.edu/bml_tube.html
This source, from the Stanford Behavioural and Neuroscience Laboratory (SBFNL)
explains the methodology of conducting a Tube Dominance Test as a means of
measuring social dominance. The dominance tube has two start-areas at each end,
in which mice of different genotypes enter. They progress towards the centre of the
tube, where they interact. The dominant mouse exhibits aggressive behaviour and
forces the submissive mouse out of the tube. Our experiment was based upon this
method, with slight alterations. Since the effects of our drug was being tested, a baseline test was initiated in order to target already aggressive mice in order to see any
change in aggression upon administration. Also, a positive control test with administration of excess serotonin was conducted to compare drug effects. The Stanford
School of Medicine is a world-renowned research intensive medical school.
(60) Garfield AS, Cowley M, Smith FM, Moorwood K, Stewart-Cox JE, Gilroy K,
et al. Distinct physiological and behavioural functions for parental alleles of imprinted Grb10. Nature. [Online] 2011;469:534-538. Available from: doi:10.1038/
nature09651 [Accessed 25th March 2012].
This study was conducted to investigate the changes in social behaviour associated
with mutant Grb10 imprinted genes in mice. Grb10 influences certain physiological
mechanisms and regulates social behaviour. The Social Dominance test was conducted between mice of various Grb10 alleles, measured through the percentage of
dominant “wins” of each mouse partaking in the dominance tube test. Our method
of graphing the experimental data from the dominance tube tests were based on the
graphs of this study, in which the percentage of “wins” was used to measure aggression. However, our data only includes the percentage of “wins” of the dominant
mouse before and after various drug administrations. Figure 3a from the study was
the basis of Figure 7 in our report.
(61) Hatayama M, Ishiguro A, Iwayama Y, Takashima N, Sakoori K, Toyota T, et al.
Zic2 hypomorphic mutant mice as a schizophrenia model and ZIC2 mutations identified in schizophrenia patients. Scientific Reports. [Online] 2011; 1(16):1-11. Available
from: doi: 10.1038/srep00016 [Accessed 15th March 2012].
This article explains the social behavioural changes in mice with reduced Zic2 expression; a gene encoding for the zinc-finger-type transcriptional regulator. Mice
with reduced Zic2 were shown to have increased locomotor activity, cognitive dysfunctions and social behavioural abnormalities. The social dominance tube test was
conducted in order to observe the difference in social dominance between mice with
and without the reduced Zic2. Results were obtained as a percentage of dominant
“wins” in each mouse. This experimental method, as well as the method of data
analysis, was used in our experiments involving a mouse treated with BZN against
a mouse with no pre-treatment in the dominance tube. The data was measured as a
percentage of dominance in the drug-treated mouse. Figure 3c from the study was
the basis for Figure 7.
(62) Eichelman B, Barchas J. Facilitated shock-induced aggression following antidepressive medication in the rat. Pharmacology Biochemistry and Behaviour. [Online]
1975; 3(4):601-604. Available from: http://www.sciencedirect.com/science/article/
pii/009130577590180X [Accessed 22th March 2012].
This study tested the effect of two different classes of antidepressive medication on
shock-induced aggression. Rats were tested for changes in shock-induced attacks
after the administration of dibenzazepine and monoamine oxidase inhibitors. The
method of calculating aggression from this study was incorporated into the testing
for functionality in vivo. Baseline testing was conducted on randomly-paired rats for
three days, after which the average percentage of attacks/50 shocks was calculated.
The study’s results showed that all of the dibenzazepine and monoamine oxidase
inhibitor groups that were used had increased levels of shock-induced aggression in
the rats, whereas no changes were seen in the rats that were administered saline or
pargyline. Tables 1 and 2 from the study were the basis for Figure 8.
(63) Sai Infusion Technologies. Brain Infusion Cannula Kits. [Online]. Available
from: http://www.sai-infusion.com/Infusion-Pumps/Alzet-Osmotic-Pump/BrainInfusion-Cannula/Brain-Infusion-Cannula-Kits.html [Accessed 22nd March 2012].
This cannula used in our in vivo behavioural testing was purchased from Strategic
Applications Inc. The cannula is required in order for our drug agent to pass through
the blood-brain barrier and localize the drug to the orbitofrontal cortex, the target
region responsible for regulating aggression.
(64) Sourcebook of criminal justice statistics. [Online]. 2011 Sept 11 [updated 2011
Sept 11;cited 2012 Mar 20]. Available from: http://www.albany.edu/sourcebook/
pdf/t31062010.pdf
This article listed criminal statistics shared by the police forces in the United States
for years ranging from 1960 - 2010. This reference was used to determine the estimated number of violent crimes, specifically aggravated assaults and murders, that
occurred in United States during 2010. We used this information to emphasize the
need for a drug that targets wrathful behaviour while justifying our presence.
(65) Police-reported crime statistics in Canada, 2010. [Online]. 2011 July 21 [updated 2011 July 11; cited 2012 Mar 22]. Available from: http://www.statcan.gc.ca/
pub/85-002-x/2011001/article/11523-eng.pdf
This article was published by Statistics Canada and reports various crime statistics
from the year 2010. This reference was used to determine whether there was a great
demand for a cure to wrath in Canada. From this source it was deduced that there
was a great deal of violent crime that took place during 2010, specifically there was in
an increase in the the number of firearm assaults and harassments across the country. This information was needed in order to justify our presence.
(66) Grant BJ, Lukman S, Hocker HJ, Sayyah J, Brown JH, McCammon JA,
et al. Novel allosteric sites on Ras for lead generation. PLos ONE. [Online]
2011:6(10):e25711. Available from: doi:10.1371/journal.pone.0025711 [Accessed 10th
March 2012].
This article outlined the use of fragment- and grid-energy based analyses to determine possible allosteric binding sites on Ras, a class of small GTPases. It also outlines how MD trajectory analysis could be accomplished, along with the accompanying statistical analysis methodology. This study is significant, as it was on the first of
its kind to employ such a methodology on GTpase sub-class.
(67) Schrödinger. Glide. (Version 5.7) [Software] Schrödinger. LLC. New York. NY.
Available from: http://www.schrodinger.com/products/14/5. 2011.
(68) Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz DT, et al.
Glide: a new approach for rapid, accurate docking and scoring .1. Method and assessment of docking accuracy. Journal of Medicinal Chemistry. [Online] 2004;47:1739–
1749 Available from: doi:10.1021/jm0306430 [Accessed 15th March 2012]
(69) Halgren TA, Murphy RB, Friesner RA, Beard HS, Frye LL, Pollard WT, et al.
Glide: a new approach for rapid, accurate docking and scoring. 2. Enrichment factors
in database screening. Journal of Medicinal Chemistry. [Online] 2004;47:1750–
1759. Available from: doi:10.1021/jm030644s [Accessed 16th March 2012].
(70) Christopoulos A, Kenakin T. G-protein coupled receptor allosterism and complexing. Pharmacological Reviews. [Online] 2002;54(2):323-374. Available from:
doi:10.1124/pr.54.2.323 [Accessed 3rd March 2012].
This article was important in that it supplied much of the knowledge of the equilibriums associated with G-protein coupled receptors. This was critical in order to understand the background knowledge required for designing the protocol, and particularly the grouping of the stable equilibrium and MD subsets.
(71) D.E. Shaw Research. Desmond Molecular Dynamics System (Version 2.4) [Software] Schrodinger. New York. NY. Available from: http://www.schrodinger.com/
products/14/3. 2010.
(72) Schrödinger. Maestro-Desmond Interoperability Tools (Version 2.4) [Software]
Schrödinger. New York. NY. Available from: http://www.schrodinger.com/products/14/3. 2010.
(73) Bowers KJ, Chow E, Xu H, Dror R, Eastwood MP, Gregersen BA, et al. Scalable
algorithms for molecular dynamics simulations on commodity clusters. Proceedings
of the ACM/IEEE Conference on Supercomputing. Report number: 6 ,2006
(74) Shivakumar D, Williams J, Wu Y, Damm W, Shelley J, Sherman W. Prediction
of absolute solvation free energies using molecular dynamics free energy perturbation and the OPLS force field. Journal of Chemical Theory and Computation. [Online] 2010;6:1509-1519. Available from: doi:10.1021/ct900587b [Accessed 5th March
2012].
(75) Guo Z, Mohanty U, Noehre J, Sawyer TK, Sherman W, Krilov G. Probing the
α-helical structural stability of stapled p53 peptides: molecular dynamics simulations
and analysis. Chemical Biology and Drug Design. [Online] 2010;75(4):348-359.
Available from: 10.1111/j.1747-0285.2010.00951.x [Accessed 5th March 2012].
Desmond was used to integrate the MD trajectory ensemble (taking into account
the stable equilibrium ensemble) into a hybrid dynamic model that represented the
intrinsic structural flexibility of 5-HT1A.
Dock Blaster:
(76) Irwin JJ, Shoichett BK, Mysinger MM, Huang N, Colizzi F, Wassam P, et al.
Automated docking screens: a feasibility study. Journal of Medicinal Chemistry.
[Online] 2009;52(18):5712-5720. Available from: doi:10.1021/jm9006966 [Accessed
15th March 2012].
DOCK Blaster is a web-based server program that performs blind-docking highthroughput virtual screening on inputted residue targets of an uploaded protein
structure file. It draws its compounds from the ZINC database. This program was
used in our protocol to identify and score potential leads compounds for our two
target allosteric sites on 5-HT1A, and also provided characteristics of each compound
via the ZINC database.