Analogues of Arylamide Phenylpiperazine Ligands To Investigate the

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Analogs of Arylamide Phenylpiperazine Ligands to Investigate the Factors Influencing D3 Dopamine Receptor Bitropic Binding and Receptor Subtype Selectivity Hamed S Hayatshahi, Kuiying Xu, Suzy A. Griffin, Michelle Taylor, Robert H. Mach, Jin Liu, and Robert R. Luedtke ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00142 • Publication Date (Web): 16 Jul 2018 Downloaded from http://pubs.acs.org on July 18, 2018

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ACS Chemical Neuroscience

Analogs of Arylamide Phenylpiperazine Ligands to Investigate the Factors Influencing D3 Dopamine Receptor Bitropic Binding and Receptor Subtype Selectivity

Hamed S. Hayatshahi1, Kuiying Xu3, Suzy A. Griffin2, Michelle Taylor2, Robert H. Mach3, Jin Liu1,* and Robert R. Luedtke2 1

Department of Pharmaceutical Sciences, University of North Texas System College of Pharmacy, University of North Texas Health Science Center.

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Department of Pharmacology and Neuroscience, University of North Texas Health Science Center.

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Department of Radiology, Perelman School of Medicine, University of Pennsylvania.

* CORRESPONDING AUTHOR INFORMATION Jin Liu, PhD. Assistant Professor of Pharmaceutical Sciences. College of Pharmacy University of North Texas Health Science Center 3500 Camp Bowie Boulevard Fort Worth, Texas 76107 [email protected]

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ABSTRACT We have previously reported on the ability of arylamide phenylpiperazines to bind selectively to the D3 versus the D2 dopamine receptor subtype. For these studies we used LS-3134 as the prototypic arylamide phenylpiperazine ligand because it binds with high affinity at D3 dopamine receptor (0.17 nM) and exhibits >150-fold D3 vs. D2 receptor binding selectivity. Our goal was to investigate how the composition and size of the nonaromatic ring structure at the piperazine position of substituted phenylpiperazine analogs might influence binding affinity at the human D2 and D3 dopamine receptors. Two factors were identified as being important for determining the binding affinity of bitropic arylamide phenylpiperazines at the dopamine D3 receptor subtype. One factor was the strength of the salt bridge between the highly conserved residue Asp3.32 with the protonated nitrogen of the nonaromatic ring at the piperazine position. The second factor was the configuration of the unbound ligand in an aqueous solution. These two factors were found to be related to the logarithm of the affinities using a simple correlation model, which could be useful when designing high affinity subtype selective bitropic ligands. While this model is based upon the interaction of arylamide phenylpiperazines with the D2 and D3 D2-like dopamine receptor subtypes, it provides insights into the complexity of the factors that define a bitropic mode of the binding at GPCRs.

Key Words Bitropic ligands D2-like dopamine receptors D3 dopamine receptor subtype Docking G-protein coupled receptor (GPCR) Molecular Dynamics Receptor subtype selective ligands Umbrella sampling

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1.

INTRODUCTION

Neurological and neuropsychiatric disorders are thought to involve complex dopaminergic disturbances in the CNS.1 For example, while dopaminergic neuron loss in the nigrostriatal pathway plays a role in Parkinson’s Disease, the nigrostriatal pathway is also thought to be involved in cognition,2 reward and craving.3 The mesocorticolimbic pathway is part of the CNS reward system, where dopamine release in the nucleus accumbens is pivotal in the expression of reward seeking behaviors. The mesolimbic pathway is involved in motivation and reinforcement, which plays a central role in the abuse of psychostimulants. The mesocortico branch of the mesocorticolimbic pathway plays a role in emotional responses related to the inappropriate affect associated with schizophrenia. Blockade of dopamine release in the tuberoinfundibular pathway can cause abnormal lactation in both males and females.4 Although the highest levels of dopamine are in the brain, dopamine also plays important physiological roles in the periphery. For example, dopamine administration increases heart rate, cardiac contractility, renal blood flow and sodium excretion.5 Dopamine receptors are members of the G-protein coupled receptor (GPCR) protein superfamily. There are two classes of dopamine receptor subtypes. The D1-like receptor subtypes include D1 (D1a) and the D5 (D1b) dopamine receptors. The D2-like receptor subtypes include D2, D3 and D4 receptors. D1-like and D2-like receptors are categorized based upon amino acid homology and pharmacological properties. For example, D1-like receptor agonists increase cAMP production whereas D2-like receptor activation inhibits forskolindependent stimulation of adenylyl cyclase.6 Dopamine receptors have been characterized using pharmacological7 and molecular biological techniques.8 Autoradiographic studies defined the neuroanatomical patterns of receptor expression in rats,9 monkeys10 and humans.11 These studies indicated that dopamine receptors are expressed in the basal ganglia, nucleus accumbens, olfactory tubercle, cingulate cortex and the prefrontal area, which are brain regions known to contain dopaminergic projections.12 Autoradiography studies indicated that the magnitude of D3 receptor expression differs from D2 receptors in the brain and that neuroanatomical localization of D2 and D3 receptor mRNA differs.13 This communication describes part of our studies to identify D3 vs. D2 dopamine receptor selective ligands that could be used a) to pharmacologically dissect the physiological role of these two dopamine receptor subtypes,14 b) as pharmacotherapeutic agents for the treatment of psychostimulant abuse15 and c) to develop positron emission tomography (PET) imaging agents that could be used to study the dynamics of D2-like dopamine receptor expression in vivo.16 Identifying highly selective D3 vs. D2 receptor selective compounds has been difficult because of the high percentage (approximately 75%) of amino acid sequence homology between the D2 and D3 receptor subtypes within the transmembrane spanning regions, which construct the orthosteric binding site of both receptor subtypes. The template for these ligands includes a substituted phenylpiperazine moiety adjacent to a four carbon chain, adjoined to an arylamide group,17 The template for our D3 dopamine receptor selective ligands includes a substituted phenylpiperazine moiety adjacent to a four carbon chain, adjoined to an arylamide group.17 We therefore describe these ligands as bitropic ligands based on the bitripoc definition by Lane et al18 as "a single ligand capable of concomitant engagement of a single receptor protein at both a primary (orthosteric) and a secondary (allosteric) binding site.”

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LS-3-134 (compound 1 in this communication), a phenylthiophene arylamide with a saturated four-carbon chain adjacent to an ortho fluoroethoxy substituted phenylpiperazine, exhibits a) high affinity binding (Ki = 0.17 nM) at human D3 receptors, b) >150-fold D3 vs. D2 dopamine receptor binding selectivity and c) low affinity binding at sigma 1 and sigma 2 receptors. Using an adenylyl cyclase inhibition assay, compound 1 was found to be a weak partial agonist at the D3 dopamine receptor (35% of maximum efficacy). These pharmacologic properties make compound 1 a good candidate for use as an 18F-labeled D3 dopamine receptor selective PET imaging agent.16b Compound 1 exhibits the highest affinity at human D3 dopamine receptors of the arylamide phenylpiperazine analogs we have evaluated. We reported on the characterization of a [3H]-radiolabeled analog of compound 1 which binds with high affinity at human (Kd = 60 pM) and rat (Kd = 200 pM) D3 dopamine receptors.19 Compound 1 is capable of a) dose-dependently reducing cocaine-induced, but not spontaneous, locomotor activity in rodents and b) reducing cocaine self-administration on a progressive ratio schedule, suggesting that compound 1 attenuates motivation for cocaine seeking behaviors (unpublished data). These findings indicate that compound 1 a) exhibits pharmacological properties similar to the previously investigated D3 dopamine receptor selective partial agonist OS-3-10615b and b) may be useful as an pharmacotherapeutic agent for the management/treatment of psychostimulant abuse. In this communication we initially focus on the importance of bulk at the piperazine position of the phenylpiperazine group in ligand affinity as it occupies the orthosteric binding site of the D2 and D3 dopamine receptor. We examined the effect of substituting the piperazine moiety with a homopiperazine, tropane, diazabicyclooctane or diazadicyclononane group upon binding at human D2 and D3 dopamine receptors. We also used computational strategies to investigate the molecular parameters associated with the binding of bitropic ligands at the D3 dopamine receptor subtype. 2. RESULTS AND DISCUSSION 2.1 Radioligand Binding Studies The chemical structure of compound 1 (previously referred to as LS-3-134,19 compound 2 and compound 3 (that correspond to the two structural segments of compound 1) are shown in Figure 1. The Ki values for these compounds at the human D2 and D3 dopamine receptor subtypes were determined using competitive radioligand binding techniques (Figures 1 and 2). This data provides empirical evidence indicating that the substituted phenylpiperazine portion of compound 1(likely occupies the orthosteric binding site of the D3 receptor while the arylamide is interacting with a secondary site on the D3 dopamine receptor protein.20 Thus, the binding mode of compound 1at the D3 dopamine receptor appears to be bitropic, where a single ligand is capable of concomitant engagement of a single receptor protein at both a primary (orthosteric) and a secondary (allosteric) binding site.18 Our previous studies indicate that the composition of the arylamide moiety of a structurally similar panel of arylamide phenylpiperazines influences both D3 vs. D2 receptor binding selectivity and efficacy.21 The phenylpiperazine compound 2 binds the D2 and D3 receptor with similar affinity (Ki values of 20-30 nM). However, since compound 1, the arylamide phenylpiperazine analog, binds with 86.5-fold greater affinity than compound 2 at the D3 dopamine receptor subtype (Figure 2), the arylamide segment of compound 1 clearly contributes to the energetics of the binding at the D3, but not the D2, receptor subtype. Therefore, our binding data indicates that

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compound 1 does not engage in a bitropic mode of binding at the D2 dopamine receptor subtype because compound 1 and compound 2 bind with similar affinity. While the substituted phenylpiperazine moiety of compound 1 provides the dominant binding energy, the arylamide portion (compound 3) of compound 1 clearly contributes to the binding energy at D3 receptors and influences D3 vs. D2 receptor binding selectivity. We then proceeded to investigate the contribution of the piperazine moiety in the phenylpiperazine group to dopamine receptor binding affinity by synthesizing a panel of analogs of compound 1 where the piperazine moiety was substituted with either a homopiperazine (compound 4), a tropane (compound 5 and compound 6) or a diazabicyclooctane (compound 7) group (Figure 3). Our binding studies indicate that as the size of the substitutent increased, the binding affinity at the D3 receptor subtype decreased. Concurrently, a series of corresponding orthosteric analogs of the aforementioned compounds were synthesized and evaluated for affinity at D2 and D3 receptors, including compound 8, compound 9, compound 10 and compound 11 (Figure 4). As anticipated, all of these compounds bound with substantially lower affinity at the D3 receptor compared to the corresponding arylamide phenylpiperazine analog (Figure 3). These orthosteric analogs had a similar progression of decreased affinity at both D2 and D3 dopamine receptors as a function of the increased size of the substituent (Figure 3 and 4). We then performed computer-assisted molecular docking, molecular dynamic (MD) and umbrella sampling simulations to better define the reason(s) for the observed decrease in affinity. 2.2.1. The bulk of the piperazine ring decreases the electrostatic interaction between the ligand and Asp3.32. At physiological pH the piperazine, homopiperazine, tropane, diazabicyclooctane and diazadicyclononane rings of the compounds evaluated in this study would be expected to be protonated at the nitrogen atom adjacent to the four carbon chain linker. Therefore, protonated ligands were used for all computational simulations. The docking of the protonated ligands to the D3 receptor crystal structure was performed using Autodock vina.22 We observed that the three highest affinity ligands in this series, compound 1, compound 2 and 4, docked to the D3 receptor (Figure 5) in a pose which included a salt bridge with aspartic acid residue 110 (Asp3.32 using the Ballesteros–Weinstein GPCR amino acid residue numbering system).23 This pose, which was found to be the best predicted pose for compound 1 had an estimated binding energy of -8.5kcal/mol. Analogous poses for compound 2 and compound 4 resulted in estimated binding energies of -6.3 kcal/mol and -8.3 kcal/mole, respectively. Alternative poses included a) bent ligands bound at the entry (extracellular side) of the receptor binding site and b) extended ligands that were unable to participate in a salt bridge with Asp3.32. It should be noted that the high affinity D2-like receptor antagonists eticlopride, which was used to crystalize the D3 receptor (PDB code 3PBL), was found to engage in a salt bridge with Asp3.32.24 Therefore, we only considered the ligand docking poses which included this salt bridge. To investigate the effect of the piperazine ring substitutions on salt bridge formation, we performed a simulation of the binding of compound 1 to the D3 receptor as predicted by the best docking pose. We performed three 300 ns MD simulations. During these simulations compound 1 remained in the orthosteric site and the salt bridge was maintained. We simulated the complexes of other four compounds in the same way as for compound 1. For a fair comparison of the interactions between different ligands and the D3 receptor, we initiated simulations of other ligand-receptor complexes from superimposed poses on compound 1. This would rule out

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the potential effect of different initial poses on the results. It is fair to assume that the other four arylamide phenylpiperazine analogs (shown in Figure 3) bind the D3 receptor in an analogous manner as compound 1. This is because these compounds share exactly the same allosteric pharmacophores and the linkers, as well as the fairly similar orthosteric pharmacophores, and they presumably make a salt bridge with Asp3.32 as all other D3 receptor binding compounds. The stability of the other ligands in the pocket was different from that of compound 1 during the simulation. We counted the number of contacts between the ligand and the protein (where a distance of < 3.0Å between atoms was considered to constitute contact) during each simulation. There was no significant difference between ligand-receptor contacts with other residues of the D3 receptor except for Asp3.32. Table 1 shows the number of the ligand contacts with Asp3.32 for the 300 ns simulations. The trend for the number of contacts between the ligand and Asp3.32 correlated with the experimental affinity values for each compound, with the exception of compound 6. This result implies that increasing the bulk at the piperazine ring position results in a weakening of the strength of the salt bridge with Asp3.32. However, the anomalous result observed for the tropane containing compound, compound 6, suggests that there are additional factors contributing to the loss of ligand affinity as the size of the substituent at the piperazine postion is increased. Molecular Mechanics-Poisson Bolzmann Surface Area (MM-PBSA) calculations25 were then performed to investigate what interaction between Asp3.32 and the ligands was affected by the substitution of the piperazine ring. The calculated binding energies and the electrostatic contribution of Asp3.32 to the total binding energy are shown in Table 1. The trend of the calculated binding energies using MM-PBSA is consistent with the experimental data, with the exception of compound 6. The Asp3.32 ligand contact number and Asp3.32 contribution to MM-PBSA binding energy shows the same trend, demonstrating that increasing the bulkiness of the piperazine ring affects binding affinities by decreasing the strength of the electrostatic interaction with Asp3.32. To further evaluate the effects of bulk at the piperazine position on the strength of the electrostatic interaction between the protonated nitrogen and Asp3.32, we performed umbrella sampling simulations for a model system in which a chloride ion is used to represent the charged aspartic acid residue. We varied the distance between the chloride ion and the protonated nitrogen of different ligands. These free energy profiles are represented as Potential of Mean Force (PMF)26 plots (Figure 6) in which the PMF values indicate how the free energy changes as a function of the distance between the chloride ion and protonated nitrogen. Accordingly, the PMF value at the local minima, of about 3.3-3.5 Å in each plot, represents the free energy of binding (∆G) of a chloride to the protonated nitrogen of the related ligand. The trend of the ∆G of binding values (Figure 6) is in general agreement with the experimental trend for the binding of these ligands to the dopamine D3 receptor reported in Table 1. This observation implies that the energetic barrier required to break a potential salt bridge is lower in ligands with lower binding affinity, demonstrating that increasing the bulk of the ring at the piperazine position weakens the ligand’s ability to form a salt bridge. Also, the local minimum in the PMF plot for compound 7 is located at longer distances relative to other ligands. This indicates that the bulky diazadicyclononane group in compound 7 likely interferes with the formation of the salt bridge. As a result, the ion locates at a greater distance away from the protonated nitrogen. However, the PMF plots indicate that compound 6 makes a slightly stronger electrostatic interaction with the ion than compound 5. While this is in variance with the experimental trend of binding affinities, it is in agreement with the number of contacts between Asp3.32 and the

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ligand’s protonated nitrogen (Table 1), as well as the electrostatic contribution of Asp3.32 to the binding affinity in the MMPBSA calculations (Table 1). This observation supports the idea that in spite of its weaker binding affinity, compound 6 makes a stronger electrostatic interaction with Asp3.32 than compound 5. Furthermore, this observation implies that a second factor is contributing to the binding affinity of these ligands at the D3 dopamine receptor. 2.2.2. Free ligand conformation is critical for D3R binding Converged MD simulations have been reported to provide a quantitative estimate of different conformations of a free molecule in solution.27 Therefore, MD simulations in explicit water (in three copies) were performed to explore the possibility that the global conformation distribution of the ligands is affected as a consequence of increasing the bulk at the piperazine ring position. One (1) µs simulation per copy was found sufficient for the simulations to be converged, except for compound 7 where the simulation was extended to two (2) µs per copy to achieve convergence. Qualitative analysis of the MD trajectories implied that the arylamide phenylpiperazine analogs exist in three distinct three-dimensional conformational ensembles: extended, folded and bent. A representative structure of each of these conformations is depicted in Figure 7. The extended, folded and bent ligand conformations can be described by the angle between three atoms, the carbon in the para position of the piperazine, the protonated nitrogen and the sulfur atom. The distributions of this angle for each ligand in MD simulations is shown in Figure 7 and its average value is reported in Table 2. The populations of ligand conformations with high values for this angle correlate with the experimentally measured affinities for D3 dopamine receptor (Figures 3 and 7). Also, the trend of the mean values agrees with the trend of experimentally measures affinities, where. the wider the angle the stronger the affinity. This agreement suggests that the compounds with a wider average angle majorly exist in extended conformation and therefore have an easier access to the orthosteric site. Conversely, compounds with the smaller average angle exist predominantly in bent conformations, which decreases their access to the orthosteric binding site. Compound 7 is an outlier in the average angle and angle distribution trends. If this were the only factor determining the binding affinity, compound 7 would be expected to have binding affinity comparable to compound 5 because their conformational distributions are similar (Figure 7). However, compound 7 has lower binding affinity than both compound 5 and compound 6. This is likely because compound 7 is not able to maintain a strong salt bridge with the Asp3.32 , as was demonstrated by our model system (Figure 6). 2.2.3. The contribution of the two major factors to the binding The results of our simulations suggest that there are two predominant factors that contribute to the affinities for the binding of arylamide phenylpiperazines to the D3 dopamine receptor: (1) the ability to form extended conformations in an aqueous solvent and (2) the ability to form a salt bridge with Asp3.32 once the ligand occupies the orthosteric binding site. The combination of these two factors is sufficient to explain the affinity outliers for each trend. The average angle between three atoms in a highly converged simulation of the free ligand (one at each end and one in the middle), shown in Figure 7, represents one factor; and the free energy of binding of a chloride ion to the ligand (Table 2) represents the second factor. Via Multiple Linear Regression (MLR), the logarithm of the affinities can be related to these factors for the studied ligands with the following equation:

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Log (affinity) = 3.007 FEBCL - 0.019 ANG + 0.675 where FEBCL is the free energy of binding of a chloride ion to the ligand and ANG is average angle of the ligand in free ligand simulations. The logarithms of affinities are estimated with this equation with coefficient of determination (r2) of 0.89 and a residual sum of squares of 0.96. This equation should be considered valid only for this series of bitropic arylamide phenylpiperazine analogs at the dopamine D3 receptor. Compounds with a different size and/or of a different chemical class might require a more (or less) complicated quantitative structureactivity relationship (QSAR) equation. Therefore, we do not expect this correlation equation to serve as a QSAR model for a variety of D3 receptor-binding compounds. However, identifying the relevant factors for this series of compounds may prove useful for the future synthetic design of bitropic D3 dopamine receptor selective ligands. The trend in the binding affinities of the orthosteric phrmacophore analogs (Figure 4) supports the contribution of the two above-mentioned factors in binding affinities. The orthosteric pharmacophore analogs are shorter than the simulated bitropic ligands and would be unlikely to exist in a compactly folded conformation. Therefore, it is expected that the conformation of the free orthosteric analogs would not have a significant effect in their binding affinity. This leaves the ability of these ligands to make a salt bridge with Asp3.32 as the only major factor contributing in binding. Accordingly, it would be reasonable to predict that compound 10 binds to the D3 receptor stronger than compound 9, unlike its bitropic counterpart compound 6, which binds to D3 receptor weaker than compound 5. This prediction, which is consistent with the experimental data (Figure 4) is based on the assumption that compound 10 and compound 6 have the same propensity for making the salt bridge with Asp3.32, as is true for compound 9 and compound 5. Therefore, in the absence of the allosteric pharmacophore and the effect of free ligand conformation in binding, it is expected that the trend of the binding affinities of the orthosteric pharmacophore analogs would follow the strength of their salt bridges with the Asp3.32. 3. DISCUSSION In our initial studies on the development of D3 dopamine receptor selective arylamide phenylpiperazine ligands we focused on evaluating the role of the aryl group on binding affinity and efficacy. We found that variations in the composition of the arylamide influenced D3 receptor affinity and D3 vs. D2 receptor binding selectivity.16a, 16b, 17, 28 It was also observed that differences in the composition of the aryl group resulted in variations in compound efficacy (from weak partial to strong partial agonist activity) at D3 receptors, as determined using an adenylyl cyclase inhibition assay. The efficacy for the same compounds for mitogenesis was consistently lower, indicating that those arylamide phenylpiperazines exhibited functional selectivity/biased agonism.21, 29 A four carbon chain connecting the arylamide and the piperazine ring was found to be optimal to maximize D3 receptor binding affinity and D3 vs. D2 binding selectivity. However, substituting a trans butylene for a saturated 4 carbon chain decreased both a) D3 vs. D2 binding selectivity and b) intrinsic efficacy.21 In our first molecular modeling study on this topic we reported that the phenylpiperazine moiety of the arylamide phenylpiperazines occupies the orthosteric binding site of both the D2 and D3 dopamine receptors. However, the orthosteric binding cavity of the D2 receptor was

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found to be shallower than the D3 receptor and the D3 receptor orthosteric site was found to be 30-50% larger than for the D2 dopamine receptor. Therefore, arylamide phenylpiperazines penetrate deeper into the D3 receptor protein and adopt a more extended/linear conformation.20a That model is consistent with a subsequent study that utilized a combination of ligand deconstruction, computational binding simulations and radioligand binding techniques,20b which indicated that the phenylpiperazine contributes the major portion of the binding energy associated with the binding of arylamide phenylpiperazines at both D2 and D3 receptors. However, the fact that the binding of the phenylpiperazine pharmacophore alone did not demonstrate D2-like dopamine receptor subtype binding selectivity again indicated the importance of the arylamide moiety in conferring D3 vs. D2 receptor binding selectivity. Subsequent studies by Peng et al.20c suggested that the benzamide moiety of the arylamide may form a hydrogen bond with a carbonyl group of C181. C181 is a highly conserved cysteine residue, which is involved in the formation of a disulfide bond between EL2 and the third transmembrane spanning helix. This disulfide bond plays an important role in the stabilization of the GPCR structure. Consequently, the orthosteric pharmacophore is anchored at the binding site and the arylamide extends toward the extracellular opening of the binding pocket, which is crucial for high D3 receptor binding affinity binding observed for the bitropic ligands such as compound 1.24 For the studies in the communication we used compound 1 as the prototypic arylamide phenylpiperazine ligand because, of the hundreds of arylamide substituted phenylpiperazine compounds that we have evaluated, compound 1was found to bind with the highest affinity at D3 dopamine receptors with >150-fold D3 vs. D2 receptor binding selectivity. Since compounds 1 and 2 bind with similar affinity at the D2 dopamine receptor and compound 1 binds with substantially higher affinity at the D3 receptor compared to the D2 receptor subtype, our first conclusion is that compound 1 engages in a bitropic mode of binding at the D3 receptor but with a monovalent mode of binding at the D2 dopamine receptor subtype. This observed enhanced binding affinity at the D3 receptor occurs despite the fact that the arylamide portion of the compound binds with very low affinity in the absence of the orthosteric pharmacophore. Therefore, the tethering of compound 1 via the orthosteric pharmacophore stabilizes the interaction of the phenylthiophene amide with a secondary site on the D3 dopamine receptor. This enhanced affinity, due to a bitropic ligand mode of engagement, has been proposed to involve the displacement of high energy water molecules bound at the secondary site that are released into the aqueous compartment, providing a favorable entropy contribution for the binding process.20b, 30 Also more evidence that these ligands engage in bitropic mode of binding comes from previous studies: The arylamide moiety, by itself, binds with very low affinity at the secondary binding site.35 The arylamide plays a role in determining D3 receptor affinity, D3 receptor efficacy and D2 vs. D3 receptor subtype binding selectivity.38 However, one might argue that the secondary binding site is not a real “allosteric” site, or even a binding pocket as it is classically defined in proteins. But it is clear that the arylamide moiety of the ligands bind to some part of the receptor causing the affinity to increase. As an allosteric site is commonly referred as a distant site31 or a distinct site from the orthosteric site32, we would argue that the term “bitropic” does describe the observed mode of binding interaction when our ligands interacts with the D3 dopamine receptor subtype, as defined by Lane and colleagues.18 The secondary binding site may not conform to the conventional or “classic” notion of a ligand binding site. Rather, it may represent a novel type of interaction potentiated by the orthosteric site but it does conform to the definition of a bitropic mode in ligand-protein interaction.

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Our initial goal for this study was to investigate how the composition and size of the nonaromatic ring structure at the piperazine position of substituted phenylpiperazine analogs might influence binding affinity at the human D2 and D3 dopamine receptors. We previously published that substitution of an aryl amide phenylpiperazine with a phenylhomopiperazine resulted in a) decreased affinity at the D3 dopamine receptor subtype, b) decreased D3 vs. D2 receptor binding selectivity and (c) an increase in intrinsic efficacy (determined using a forskolin-dependent adenylyl cyclase inhibition assay).33 In the studies for this communication it was found that substituting the piperazine moiety of compound 1 with a homopiperazine, tropane, diazabicyclooctane or diazadicyclononane group resulted in an incremental decrease in binding affinity at both D2 and D3 receptors as a function of the increased size of these nonaromatic ring substituents. The availability of coordinates for the three-dimensional structure of the D3 dopamine receptor subtype enabled us to use computational techniques to investigate the molecular basis for loss of binding affinity as the size of the ring at the piperazine position increased. The computational ligand docking studies indicated the importance of the ability of a protonated nitrogen in these rings to engage in an electrostatic interaction with an Asp3.32 residue located in the third helical transmembrane spanning region. This aspartate has been found to be highly conserved in monoamine GPCRs, including all of the D1-like and D2-like dopamine receptor subtypes (subtypes 1-5), the beta 1- and beta 2-adrenergic receptors and the serotonin GPCR receptors. The importance of this residue for binding adrenergic agonists has been verified by site directed mutagenesis studies, as well as for somatostatin and opioid receptors.34 Furthermore, evidence for the importance of these residues in stabilizing D2-like dopaminergic antagonist binding was obtained from the crystallographic analysis of the D3 dopamine receptor where it was found that a protonated nitrogen on the high affinity antagonist eticlopride, which was used to stabilize the D3 receptor crystal structure, engaged in a salt bridge with Asp3.32.24 Ligand-protein complex simulations for the interaction of the D3 receptor with our synthetic analogs indicated that the orthosteric binding site is large enough to accommodate the analogs with bulkier rings at the piperazine position. However, the bulkier ring decreases the frequency of the contact of the ligand to form the salt bridge with Asp3.32. This decrease is also accompanied by a decrease in the binding affinity and the electrostatic contribution of Asp3.32 to the binding. In these complex MD simulations, we also found that while the number of ligand contacts with Asp3.32 varied, contacts with residues other than Asp3.32 did not differ significantly. This data provides additional evidence for the detrimental effect of the piperazine ring modifications on the strength of the salt bridges with Asp3.32. Interestingly, compound 6 was found to be an outlier in this trend because it makes more contacts with Asp3.32 than compound 5 or compound 4 and its electrostatic interaction with Asp3.32 was found to be stronger. Additionally, the total estimated binding energy for compound 6 is greater. This observation implied that if compound 6 has the same opportunity to bind as compound 5 and compound 4, it should bind with greater energy. This anomaly suggested that there was likely an additional factor or factors contributing to the energetics profile of compounds 1,4,5,6 and 7. This trend of electrostatic contributions was examined using a model system in which the free energy profile was calculated as a function of increasing the distance of a chloride ion from a protonated nitrogen. These results were found to be in close correlation with the number of contacts of ligands with Asp3.32 and its calculated electrostatic contributions to the MMP-BSA binding energies. This relative correlation shows that we can use such simple, cheap and fast

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model to speculate the capability of these compounds for making the salt bridge with Asp3.32 without performing the costlier simulation of the ligand-receptor complex. Subsequent MD simulations provided an insight into one additional factor. These simulations indicated that the composition of the piperazine substitution affects the distribution of the free, unbound ligand conformations in the aqueous compartment. The arylamide phenylpiperazine analogs appear to exist in three predominate configurations: folded, bent and extended conformations. For ligands where a more compact, folded conformation predominates, there was a correlation with lower binding affinity at the D3 receptor. Compound 7 was found to be an outlier in this correlation because while its conformation distribution is very similar to that of compound 5, it forms a relatively weak electrostatic interaction with Asp3.32 and therefore, its affinity is lower. More compact ligand conformation in the aqueous compartment presumably increase the energy cost for adopting the bound conformation by the ligand. In this work, we developed a simple, fast and cheap MD simulation approach that would relatively and qualitatively represent this energy cost, which could be more precisely calculated with costly simulations that involve the presence of the receptor. We predict that this simple approach can be utilized for filtering out the ligands that need high energy cost for conformational change without the need to perform expensive ligand-receptor complex simulations. 4. CONCLUSIONS In summary, two major factors were identified as being important for determining the binding affinity of bitropic arylamide phenylpiperazines at dopamine D3 receptor subtypes. One factor was the strength of the salt bridge between the highly conserved residue Asp3.32 with the protonated nitrogen of the nonaromatic ring at the piperazine position. The second factor relates to the conformation of the bitropic unbound ligand in an aqueous solution. These factors were found to be related to the logarithm of the affinities using a simple correlation model with Multiple Linear Regression (MLR). The distortion of the trend of experimental affinities of the orthosteric pharmacophores relative to their bitropic counterparts further supports the importance of these two factors because the orthosteric pharmacophores are not likely to form the compactly folded conformations, which rules out the effect of the free ligand conformation on binding. These studies provide insights into the complexity of the physicochemical and molecular pharmacological factors that define the modes of binding of bitropic ligands at GPCRs. While the model presented in this manuscript is based upon the interaction of arylamide phenylpiperazines with D2-like dopamine receptors, it provides a) insights into the complexity of the factors that define bitropic modes of the binding for extended ligands and b) a generalized correlation model that could be used when designing novel high affinity subtype selective bitropic ligands. The modeling approaches presented in this manuscript are designed in a way to provide valuable predictive information with computationally low-cost simulations. Here, we show that fast conventional MD simulations and umbrella sampling simulations of unbound ligands provide us with a reasonable explanation of the trend of the experimental binding affinities of a small subset of bitropic arylamide phenylpiperazines. Application of these and similar fast computational approaches is promising for drug developers who tend to filter out weakly binding compounds in a particular ligand-receptor system prior to the costly chemical synthesis of such compounds. Of course, tuning and validating of these receptor-free MD-based approaches should be investigated in future in other ligand-receptor systems.

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5. METHODS 5.1 Chemistry Reagents were purchased from Sigma-Aldrich and Fisher Scientific. Silica gel chromatography was carried out on a Biotage IsoleraTM Spektra One chromatograph system. All synthesized compounds were analyzed and confirmed to have purity over 95% with a Waters Alliance LC-MS system. NMR spectra were measured on a Brucker 500 or 360 MHz spectrometer as indicated. Chemical shifts (δ values) were reported in ppm relative to TMS. For multiplicity, s = singlet, d = doublet, t- triplet, m = multiplet. 1H NMR spectra data were presented as follows: Chemical shifts (multiplicity, coupling constants, integration). General Procedure A for butylation: The appropriate diaza compound (0.1-0.5 mmol, 1 eq) and 1-bromobutane (4 eq) were dissolved in acetonitrile (3 ml). K2CO3 (3-4 eq) was added. The mixture was kept stirring at 80°C overnight. The mixture was filtered and the filtrate was condensed. The residue was applied to FC with hexanes/ethyl acetate or dichloromethane/methanol to yield the butylamino compounds. General Procedure B for coupling: 4-(thiophen-3-yl)benzoic acid (1 eq) and HCTU (1 eq) were dissolved in dimethylformamide (2 ml) at 0°C, DIPEA (3 eq) was added. The mixture was kept stirring at 0°C for 15 min. A mixture of the appropriate amine compound (1 eq) and DIPEA (3 eq) was added at 0°C. The mixture was kept stirring at r.t. overnight. After that the reaction mixture was diluted with ethyl acetate (30 ml) and washed with water (20 ml x 2), NaHCO3 aqueous (20 ml x 2) and brine (20 ml x a). The organic layer was dried over Na2SO4 and condensed. The residue was applied to FC (hexanes/ethyl acetate or dichloromethane/methanol) to give the amide compounds. General Procedure C for arylation: The appropriate diaza compound (1 eq), 1-chloro-2-(2fluoroethoxy)benzene1 (1.2 eq), Pd2(dba)3 (0.01 eq), RuPhos (0.02 eq), NaOtBu (1.5 eq) and dioxane (3 ml/mol) were put in a sealed reaction vial. The vial was then placed in a preheated 100°C oil bath and stirred for 10 min. The solvent was removed under reduced pressure resulting in a crude oily residue. The crude product was purified by FC with dichloromethane/methanol (0-10%) or ethyl acetate/methanol (0-10%) to give the arylated compounds. General Procedure D for preparing the pthalimidobutyl compound: The free amine (1 eq) compound was dissolved in dichloromethane (3 ml/mmol), N-4-bromobutylphthalimide (1.5 eq) was added followed by the addition of trimethylamine (0.1 ml). The mixture was kept stirring at room temperature overnight. The mixture was condensed and the residue was applied to FC (dichloromethane/methanol 0-10%) yielding the pthalimidobutyl compound. General Procedure E for deprotection of the pthalimidobutyl compound.: The pthalimido compound (1 eq) was dissolved in methanol (2 ml/mmol), hydrazine hydrate (3 eq) was added. The mixture was kept stirring at 80°C overnight. The mixture was condensed and the residue was applied to FC (dichloromethane/7N methanolic ammonia, 0-20%) yielding the free amine. General procedure F for Boc deprotection: The boc protected compounds were dissolved in 2N HCl/diethyl ether and the mixture was kept stirring at room temperature overnight. The solid was filtered and washed with ether. The solid obtained was either used directly for the next step or neutralized with 7N ammonia/methanol then purified with FC (dichloromethane/7N methanolic ammonia) to yield the free amine. 5.2 Transfection, tissue culture and cell homogenate preparation.

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Stably transfected HEK293 cells expressing human D2 or D3 dopamine receptors were prepared using a pIRESneo2 expression vector. Transfected cells were maintained in Dulbecco’s modified Eagle’s medium–high glucose media with 10% fetal calf serum (Invitrogen, Carlsbad, CA, USA) with penicillin (100 units/mL), streptomycin (100 µg/ mL) and G418 (400 µg/mL G418). HEK293 cells were grown to approximately 80% confluence, harvested, and resuspended in homogenization buffer (50 mM Tris-HCl/150 mM NaCl/10 mM EDTA buffer, pH 7.5) at 4°C. The cell pellet was collected by centrifrugation and homogenized using a Polytron homogenizer (Brinkmann Instruments, Westbury, NY, USA (setting 6)). The cell homogenate was centrifuged at 12 000 g at 4°C for 15 min. This pellet was resuspended in cold homogenization buffer, kept on ice, aliquoted (0.5 mL) into 1.5-mL plastic microfuge vials [1.0 mL/T75 flask (BD Falcon, BD Biosciences, San Jose, CA, USA)], and kept at -80°C. 5.3 Competitive radioligand-binding studies. For competitive binding studies, transfected HEK293 cell homogenates were suspended in homogenization buffer and incubated with radioligand [125I]IABN, in the presence or absence of inhibitor at 37°C for 60 min with [125I]IABN (total volume = 150 µL) as previously described.35 Competitive radioligand binding studies were performed to determine the concentration of inhibitor that inhibits 50% of the specific binding of the radioligand (IC50 value). The final radioligand concentration was approximately equal to the Kd value for the binding of the radioligand. For each competition curve, triplicates were performed using two concentrations of inhibitor per decade over five orders of magnitude. Binding was terminated by the addition of cold wash buffer (10 mM Tris–HCl/150 mM NaCl, pH = 7.5) and filtration over a glass-fiber filter (Pall A/B filters, #66198). A Packard Cobra Gamma Counter was used to measure the radioactivity of [125I]IABN. The competition curves were modeled for a single binding site using

Bs = Bo - (( Bo +L)/(IC50 + L)) where Bs is the amount of ligand bound to receptor and Bo is the amount of ligand bound to receptor in the absence of competitive inhibitor. L is the concentration of the competitive inhibitor. The IC50 value is the concentration of competitive inhibitor that inhibits 50% of the total specific binding. IC50 values were determined using non-linear regression analysis with Table Curve 2D v 5.01 (Jandel, SYSTAT, Systat Software, Inc., San Jose, CA, USA). The values for Bns and Bo were constrained using experimentally derived values. The IC50 values were converted to equilibrium dissociation constants (Ki) using the Cheng and Prusoff (1973) correction.36 Mean Ki values + S.EM. are reported for at least three independent experiments. 5.4 Computational studies. 5.4.1 Docking. Autodock tools37 were used to prepare the model of the crystal structure of the D3 dopamine receptor (PDB code: 3PBL)24 as well as the ligands for docking studies. All single bonds in ligands were flexible except the amide bond, which was fixed at the trans configuration. The piperazine ring of the ligands were protonated at the nitrogen proximal to the amide bond. A search box was defined at the orthosteric binding site of the receptor with 44, 50 and 100 Å dimensions for docking the ligands with Autodock vina.22 The crystallographic ligand was docked as a benchmark to test the capability of Autodock vina to accurately predict its binding

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pose. The result docking pose was very similar to the crystallographic pose (supporting figure). Then, all other ligands were docked on the receptor using the same protocols as for the crystallographic ligand. 5.4.2 Building the free ligand systems. Multi-conformational partial charge fitting was performed using RED-III38 to prepare the ligands for molecular dynamics simulations. To do so, the conformations of the first nine docking poses of each ligand were optimized with QM energy minimization using Gaussian 09 at HF/6-31G*. Three sets of three atoms that do not occur in the same straight geometric line were used to define reorientation of the optimized structures of each ligand for RED-III. Accordingly, RESP partial charges39 were generated for ligands that were consistent with all conformations and orientations of the ligand. A random conformation of each ligand was solvated in TIP3P water model40 using tLEaP of AmberTools 16 41 with GAFF force field parameters.42 The octahedral simulation box size was chosen in a way that fits at least 1100 water molecules. One chloride ion was added to the simulation box to neutralize the protonated ligands (Table 2). 5.4.3 Building the complexes. MD simulations of the protein-ligand complexes in membrane and explicit water were performed to study the stability of the ligands in the binding site. MD simulation of a truncated transmembrane domain of the D3 dopamine receptor was performed by Sansom group 43 and is available for free download at http://memprotmd.bioch.ox.ac.uk. We used their truncated version, which contains the orthosteric site and has shown stability in their MD simulations. The CHARMM online membrane builder was used to build a membrane around the protein containing phosphatidylcholine, phosphatidylethanolamine and cholesterol with 2:2:1 ratio. 150 mM NaCl was also added with some TIP3P water residues to the simulation box. We then used the charmmlipid2amber.py program in AmberTools 16 41 to change the lipid residue names to Amber format. The atoms in ligands were also renamed with antechamber program in AmberTools 16 to match the GAFF force field names. Then the ligands were manually superimposed on the best docking pose of compound 1 in the protein orthosteric binding site in a way that the salt bridge between the protonated nitrogen in the piperazine ring and D110 was maintained. Subsequently, tLEap was used to prepare the simulation box and the topology with Amber FF14SB and Lipid FF14SB force fields. Joung-Cheatham TIP3P related ion parameters44 were used for Na+ and Clions. 5.4.4 Equilibration and production simulations, and their analysis. Solvated free ligands and ligand-protein complexes were equilibrated using the same nine step protocol as described in detail in a previous work.45 In this protocol, multiple steps of restrained minimization and MD simulations in constant volume and constant pressure were performed to ensure that all possible clashes are relaxed and the water density is equilibrated. These steps were followed by a 1 ns MD simulation with no restraints in constant pressure. The ligand-protein complex production simulations were performed for 300 ns per copy in three copies. In each copy, the positions of ions in each copy were put in new random positions using the cpptraj program of AmberTools 16.46 To facilitate MD in a 0.004 fs timescale, hydrogen mass repartitioning (transferring part of the mass of the heavy atoms to the

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hydrogen atoms that are connected to them) was performed on the system with parmed program in AmberTools 16.41 The simulations were performed using pmemd.cuda in Amber 1641 on local GPUs at 310.15 K, which was controlled by Langevine thermostat 47 at constant pressure which was regulated via Monte Carlo barostat with pressure relaxation time of 5 ps. SHAKE48 was used to constrain the bonds to the hydrogens, and particle mesh Ewald (PME)49 was used with 9.0 Å to calculate the long range interactions. The coordinates were saved onto the trajectories every 10 ps. The free ligand production simulations were also performed in the same conditions as ligand-protein complex simulations. However, the free ligand simulations were extended to 1 µs per copy in three copies except for compound 7, which was extended to 2 µs per copy. All simulations were analyzed using cpptraj program in AmberTools 16.46 5.4.5 Umbrella sampling. Accordingly, a reaction coordinate was defined as the distance between the chloride ion and the protonated nitrogen on each ligand. The topology and initial coordinates of the free ligand simulations were used for the umbrella sampling simulations, except that the chloride ion was positioned at a distance of 2.8 Å away from the protonated nitrogen. The systems were equilibrated as free ligand simulations with an additional distantance restraint with force constant of 100 kcal/mol-Å2 that would keep the ion at this distance. Then, a production simulation for 2.8 Å distance on the reaction coordinate was performed for 500 ps. The final snapshot of this simulation was used as the initial structure for a 520 ps simulation at a distance of 2.9 Å. This sequence of serial 520 ns simulations was performed until the ion was at a distance of 6.0 Å, where it is fully solvated. The first 20 ps of each umbrella simulation was considered to be at equilibrium and was not used for data collection. The production simulation was performed as the production phase of the free ligand simulations, except that hydrogen mass repartitioning was not applied and the time step was 0.002 ps. The distance between the ion and the protonated nitrogen was recorded at every 10 simulation steps, resulting in 25000 data points per umbrella. The histograms of data points for different umbrellas were checked to ensure that they reasonably overlap (supporting figure). In summary, 33 umbrella simulations were performed, each for 500 ps, with a 0.1 Å distance interval between 2.8 Å to 6.0 Å. Our simulations indicated that distances a) 6.0 Å would cause an artifact resulting from the periodic box conditions. WHAM (Weighted Histogram Analysis Method) 26, 50 that was implemented by Alan Grossfield (http://membrane.urmc.rochester.edu/sites/default/files/wham/doc.html) was used to generate the potential mean force (PMF) plots with a convergence tolerance of 10-9 at 310.15 K.

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FIGURES Figure 1. Structures of Compound 1 Plus Orthosteric and Allosteric Pharmacophores.

The chemical structure of compound 1 and two structural segments of this compound (compound 2 and compound 3) are shown. The mean Ki values + S.E.M. (n = 3) for the binding of these compounds at both human D2 and human D3 receptors is also shown.

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Figure 2. Competitive Radioligand Binding Curves for the Binding at Human D3 Dopamine Receptors. Human D2 Dopamine Receptors

Human D3 Dopamine receptorsLS Theo D3

100

100

Percent Specific Binding (%)


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


90 80 70 60 50 40 30 20 10

80 70 60 50 40 30 20 10

0

10-12

90

10-11 10-10 10-9 10-8 10-7 10-6 Compound Concentration (Molar)

10-5

0

10-12


10-5

Composite competitive radioligand binding data (n = 3) is shown for the binding of compound 1 (), compound 2 () and compound 3 () at human D2 (left panel) or D3 (right panel) dopamine receptors.

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Figure 3. Structures of Compound 1 Phenylpiperazine Analogs.

The chemical structure of four structural analogs of compound 1 (compounds 4, 5, 6 and 7) are shown. The mean Ki values + S.E.M. (n = 3) for the binding of these compounds at both human D2 and human D3 receptors are also shown.

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Figure 4. Structures of Orthosteric Pharmacophore Analogs.

The chemical structures of four orthosteric pharmacophore ligands (compound 8, 9, 10 and 11) are shown. The mean Ki values + S.E.M. (n >3) for the binding of these compounds at both human D2 and human D3 receptors is also shown.

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Figure 5. Docking of Compounds 1, 2 and 4 to the Human D3 Dopamine Receptor.

Docking of compound 1 (top), compound 2 (middle) and compound 4 (bottom) to the human D3 dopamine receptor is shown.

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Figure 6. The Free Energy Profiles in Terms of Potential Mean Forces (PMF) for Increasing the Distance Between a Chloride Ion and the Protonated Nitrogen of Compounds.

The PMF plots for increasing the distance of a chloride ion from the protonated nitrogen of compounds 1, 2, 4, 5, 6 and 7. The values at local minima (2.8 Å-3.0 Å) represent the bound ion, while the values close to 6.0 Å represent the fully-solvated ion, and the difference between these values represents the free energy for binding of the ion to the protonated nitrogen.

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Figure 7. Summary of the Results of Molecular Dynamic Simulation Studies to Estimate the Conformations of Unbound Ligands in an Aqueous Solution.

(Top) The sample structures of folded, bent and extended conformations (left to right) of compound 7. (Bottom) Distribution histograms of the described angle (see text) in free ligand simulations averaged over three runs of 1 µs length (2 µs for compound 7). Error bars represent the standard deviation between three runs.

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TABLES

Table 1. Summary of Binding Simulations and MM-PBSA Calculations.

Ligands compound 1 compound 4 compound 5 compound 6 compound 7

Affinity (nM) 0.17 ± 0.01 19.4 ± 1.10 34.8 ± 1.10 115 ± 23.10 1,644 ± 223.00

Mean Contact Number with Asp3.32 28.99 ± 0.80 29.91 ± 10.31 14.41 ± 13.07 26.09 ± 1.60 6.95 ± 9.65

Asp3.32 –Ligand Calculated Binding Energy (kcal/mol) using (MM-PBSA) -3.54 ± 2.95 -0.45 ± 6.43 +0.34 ± 3.96 -3.13 ± 3.06 +5.21 ± 4.42

Electrostatic Contribution of Asp3.32 to MM-PBSA energy (kcal/mol) -45.79 ± 0.60 -42.39 ± 3.61 41.81 ± 3.26 -43.70 ± 0.46 -38.74 ± 0.60

The average number of contacts of nitrogen atoms in ligands in complex simulations with oxygen atoms in Asp3.32 with a < 3.0 Å cutoff divided by 1000, their MM-PBSA binding energies on the initial 10 ns of the simulations and the electrostatic contribution of Asp3.32 interactions to the MM-PBSA binding energies, averaged over three runs is shown. The binding affinity was obtained from competitive radioligand binding studies. The errors are standard deviations between three runs.

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Table 2. Comparison of Ligand Affinity, Mean Folding Angle and the Free energy of binding to a chloride ion (PMF values). Free energy of binding to a chloride ion Mean Angle Binding Affinity Ligand (kcal/mol) (degrees) (nM) 111.5 ± 1.6 0.17 ± 0.01 0.34 ± 0.16 compound 1 98.5 ± 1.7 19.4 ± 1.1 0.61 ± 0.21 compound 4 82.5 ± 0.5 34.8 ± 1.1 1.02 ± 0.34 compound 5 115 ± 23.1 76.8 ± 0.31 0.89 ± 0.20 compound 6 86.9 ± 1.1 1644 ± 233 1.36 ± 0.15 compound 7 The Ki values obtained from radioligand binding studies and the mean folding angle for the five (5) phenylpiperazine aryamide anlaogs are shown. The angle values are calculated from the MD simulations of the free ligands and the error values are standard deviations between three runs. The free energies of binding to a chloride ion are calculated based on the PMF plots in Figure 6.

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Figure Legends. Figure 1. Structures of Compound 1 Plus Orthosteric and Allosteric Pharmacophores. The chemical structure of compound 1, compound 2 and compound 3 are shown. The mean Ki values + S.E.M. (n = 3) for the binding of these compounds at both human D2 and human D3 receptors is also shown. Figure 2. Competitive Radioligand Binding Curves for the Binding at Human D3 Dopamine Receptors. Composite competitive radioligand binding data (n = 3) is shown for the binding of compound 1 ( ), compound 2 ( ) and compound 3 ( ) at human D2 (left panel) or D3 (right panel) dopamine receptors. Figure 3. Structures of LS-3-134 (compound 1) Phenylpiperazine Analogs. The chemical structure of four (4) structural analogs of compound 1 are shown. The mean Ki values + S.E.M. (n = 3) for the binding of these compounds at both human D2 and human D3 receptors are also shown. Figure 4. Structures of Orthosteric Pharmacophore Analogs. The chemical structure of four (4) orthosteric pharmacophore ligands is shown. The mean Ki values + S.E.M. (n >3) for the binding of these compounds at both human D2 and human D3 receptors is also shown. Figure 5. Docking of Compound 1, 2 and 4 to the Human D3 Dopamine Receptor. Docking of compounds compound 1 (top), compound 2 (middle) and compound 4 (bottom) to the human D3 dopamine receptor is shown. Figure 6. The Free Energy Profiles in Terms of Potential Mean Forces (PMF) for Increasing the Distance Between a Chloride Ion and the Protonated Nitrogen of Compounds. The PMF plots for increasing the distance of a chloride ion from the protonated nitrogen of compounds 1, 2, 4, 5, 6 and 7. The values at local minima (2.8 Å-3.0 Å) represent the bound ion, while the values close to 6.0 Å represent the fully-solvated ion, and the difference between these values represents the free energy for binding of the ion to the protonated nitrogen. Figure 7. Summary of the Results of Molecular Dynamic Simulation Studies to Estimate the Conformations of Unbound Ligands in an Aqueous Solution. (Top) Affinities for the five aryl amide phenylpiperazines discussed in this manuscript and the average angle made by three atoms on them: two at the ends and the protonated nitrogen. (Middle) The sample structures of folded, bent and extended conformations of compound 3. (Bottom) Distribution histograms of the described angle in free ligand simulations averaged over three runs of 1 µs length (2 µs for compound 3. The error bars represent the standard deviation between three runs.

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ACKNOWLEDGEMENTS Funding for these studies were provided to RRL and RHM by DA029840 and DA023957.

CORRESPONDING AUTHOR INFORMATION J. Liu, PhD. Assistant Professor of Pharmaceutical Sciences. College of Pharmacy University of North Texas Health Science Center 3500 Camp Bowie Boulevard Fort Worth, Texas 76107 [email protected]

Author Contributions H. H. performed molecular modeling studies and drafted major portions of the manuscript. J. L. performed and supervised molecular modeling studies and drafted major portions of the manuscript. S. A. G. performed binding studies, data analysis and manuscript preparation. M. T. performed binding studies, data analysis and manuscript preparation R. R. L. drafted the manuscript and supervised binding studies and data analysis. K. X. synthesized compounds used in this study R. H. M. designed the research project, supervised synthetic chemistry and drafted portions of the manuscript.

ABBREVIATIONS USED

PET CNS Kd value Ki value IC50 value GPCR

positron emission tomography central nervous system affinity association constant determined using direct binding techniques affinity association constant determined using competitive binding techniques concentration of inhibitor that inhibits 50% of the specific binding of the radioligand G Protein Coupled Receptor

SUPPORTING INFORMATION Supporting information includes a file containing details of the compounds synthesis and two figures about docking and umbrella sampling computations.

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Graphic Abstract

67 D2 67 D3 65 D3

D3 Data 1

D3 Dopamine Receptors

100 90

65 D2

100

80 70 60 50 40 30 20 10 0

Folded

Bent

Extended

10

-12

10

-11

-10

-9

-8

-7

-6

10 10 10 10 10 Compound Concentration (Molar)


D2 Data 1

LS D3 65 theo D3 67 Theo D3 LS Theo D3


Conformations of Unbound Ligands


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 32 of 32

10

-5

LS D2 65 Theoretical LS Theoretical 67 theoretical

D2 Dopamine Receptors

90 80 70 60 50 40 30 20 10 0

10-12


10-5

Analogues of Arylamide Phenylpiperazine Ligands To Investigate the

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