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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.