Current drug treatments targeting dopamine D3 receptor

    Current drug treatments targeting dopamine D3 receptor Gian Marco Leggio, Claudio Bucolo, Chiara Bianca Maria Platania, Salvatore Salomone, Filippo Drago PII: DOI: Reference:

S0163-7258(16)30097-3 doi: 10.1016/j.pharmthera.2016.06.007 JPT 6919

To appear in:

Pharmacology and Therapeutics

Please cite this article as: Leggio, G.M., Bucolo, C., Platania, C.B.M., Salomone, S. & Drago, F., Current drug treatments targeting dopamine D3 receptor, Pharmacology and Therapeutics (2016), doi: 10.1016/j.pharmthera.2016.06.007

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ACCEPTED MANUSCRIPT P&T 22933

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Current drug treatments targeting dopamine D3 receptor

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Gian Marco Leggio, Claudio Bucolo, Chiara Bianca Maria Platania, Salvatore Salomone*,

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Filippo Drago

Department of Biomedical and Biotechnological Sciences, School of Medicine, Catania

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University, Catania, Italy

Corresponding author:

Salvatore Salomone, M.D., Ph.D.

Department of Biomedical and Biotechnological Sciences Catania University

Viale Andrea Doria 6, 95125 Catania, Italy. Tel 39-095-7384085 Fax 39-095-7384228 E-mail: [email protected]

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Running title: D3R-targeting drugs

ACCEPTED MANUSCRIPT Abstract Dopamine receptors (DR) have been extensively studied, but only in recent years they

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became object of investigation to elucidate the specific role of different subtypes (D1R, D2R,

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D3R, D4R, D5R) in neural transmission and circuitry. D1-like receptors (D1R and D5R) and D2-like receptors (D2R, D2R and D4R) differ in signal transduction, binding profile,

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localization in the central nervous system and physiological effects. D3R is involved in a

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number of pathological conditions, including schizophrenia, Parkinson’s disease, addiction, anxiety, depression and glaucoma. Development of selective D3R ligands has been so far

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challenging, due to the high sequence identity and homology shared by D2R and D3R. As a consequence, despite a rational design of selective DR ligands has been carried out, none of

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currently available medicines selectively target a given D2-like receptor subtype. The

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availability of the D3R ligand [11C]-(+)-PHNO for positron emission tomography studies in

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animal models as well as in humans, allows researchers to estimate the expression of D3R in vivo; displacement of [11C]-(+)-PHNO binding by concurrent drug treatments is used to estimate the in vivo occupancy of D3R. Here we provide an overview of studies indicating D3R as a target for pharmacological therapy, and a review of market approved drugs endowed with significant affinity at D3R that are used to treat disorders where D3R plays a relevant role.

Key words: dopamine D3 receptor, aripiprazole, blonanserin, buspirone, cabergoline, cariprazine

ACCEPTED MANUSCRIPT Contents 1. Introduction

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2. Diseases involving D3R transmission

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3. Rational design of selective D3R ligands 4. In vivo imaging of D3R

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5. Drug treatments targeting D3R

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6. Concluding Remarks Conflict of interest statement

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Acknowledgments

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References

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Abbreviations: 5-HT, 5-hydroxytryptamine; 7-OH-DPAT, 7-hydroxy-N,N-di-n-propyl-2aminotetralin; [11C]-(+)-PHNO, [11C]-(+)-propyl-hexahydro-naphtho-oxazin; AH, aqueous humor; BDNF, Brain-Derived Neurotrophic Factor; CNS, central nervous system; DA, dopamine; DR, dopamine receptor; D2LR, dopamine receptor 2 long form splice variant; D2SR, dopamine receptor 2 short form splice variant; D3R-/-, dopamine receptor 3 null mice; EMA, European Medicines Agency; FDA, Food and Drug Administration; IOP, intraocular pressure; L-DOPA, L-3,4-dihydroxyphenylalanine; LID, L-DOPA-induced dyskinesia; NOR, novel object recognition; PD, Parkinson’s disease; PET, positron emission tomography; WT, wild type; SAR, structure activity relationship; SUD, Substance Use Disorder.

ACCEPTED MANUSCRIPT 1.Introduction Dopamine (DA) activity in the central nervous system (CNS) is mediated by five G protein-

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coupled receptors grouped in two classes, the D1-like receptors (D1R and D5R) and the D2-

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like receptors (D2R, D2R and D4R), which differ in their signal transduction, binding profile and physiological effects (Beaulieu & Gainetdinov, 2011; Seeman & Van Tol, 1994). D1-like

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receptors are principally coupled to stimulatory Gs-proteins and enhance the activity of

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adenylyl cyclase, whereas D2-like receptors are primarily coupled to inhibitory Gi-proteins and suppress the activity of adenylyl cyclase (Beaulieu & Gainetdinov, 2011). D2-like receptors

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represent the most relevant class in the pathophysiology of neurological and psychiatric disorders. However, while the role of the D2R subtype has been extensively studied in

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several neuropsychiatric diseases such as schizophrenia, Parkinson’s disease (PD) and

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addiction, the role of D3R and D4R is still under investigation. The primary sequence of D3R

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is close to that of the D2R and, to a lesser extent, of D4R. Human D2R and D3R are highly homologous (Sibley & Monsma, 1992), sharing 78% of sequence identity in the transmembrane domains, including the binding site (Shi & Javitch, 2002). This sequence identity has introduced difficulties in the design of selective ligands. The initial lack of selective pharmacological tools delayed the elucidation of the role of D3R and raised even questions about the physiological significance of the D3R. From the beginning, attention has been attracted to the restricted distribution of the D3R in brain. Indeed, the D3R subtype shows a distinct distribution with high levels in the limbic system, including the islands of Calleja, the nucleus accumbens and the olfactory tubercles, brain areas critically involved in the regulation of motivation, reward and cognitive functions (Landwehrmeyer et al., 1993; Levesque et al., 1992). Available evidence indicates that D3R functions, at least in part, as an autoreceptor (i.e. a presynaptic receptor), with inhibitory effects on DA impulse flow and DA

ACCEPTED MANUSCRIPT release (Gobert et al., 1995; Tepper et al., 1997). It has been shown that D3R-null mice (D3R/-

) have extracellular levels of DA twice as high as their wild-type (WT) littermates, consistent

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with the presynaptic location mentioned above, where D3R would inhibit DA release (Joseph et al., 2002; Koeltzow et al., 1998).

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As mentioned above, D3R activates Gαi/o proteins to inhibit cAMP production and

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decrease protein kinase A (PKA) activity (Missale et al., 1998; Robinson and Caron, 1997),

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but D3R also regulates other intracellular pathways, including the extracellular signal regulated kinase 1/2 and Akt cascades through G protein-dependent and/or independent

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mechanism, this latter involves β-arrestin (Collo et al., 2008, 2012; Cussac et al., 1999). The ability of ligands to differentially affect signaling through these pathways, referred to as biased

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agonism or functional selectivity, may be therapeutically exploitable. Recently, ligands that

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are devoid of D2R-mediated Gαi/o protein signaling, but behave as partial agonists for D2R/β-

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arrestin interactions, have been found to exert a number of effects in preclinical models of schizophrenia-like behavior while causing lower catalepsy (Park et al., 2016). To the best of our knowledge, biased agonism of D3R selective antipsychotics has not yet been documented, but we may predict that, as reported for D2R, it could support fewer side effects and greater therapeutic efficacy for treating conditions such as schizophrenia. As the majority of G protein-coupled receptors, D3R forms both homo- and heteromers (recently reviewed by Maggio et al., 2015). Heteromers have been reported with D2R (Scarselli et al., 2001), D1R (Fiorentini et al., 2008; Marcellino et al., 2008), and also with the adenosine receptor A2AR (Torvinen et al., 2005). DR heteromers exhibit pharmacological and cell signaling properties distinct from their constituent receptors (Lee et al., 2004). Binding to D2R-D3R heteromers may account, at least in part, for the antipsychotic effect of aripiprazole and N-desmethylclozapine (Novi et al. 2007). In fact, while these two compounds behave as

ACCEPTED MANUSCRIPT partial agonists at D2R, they behave as antagonists at the D2R-D3R heteromers. On the other hand, activation of D1R–D3R heteromers is putatively involved in L-DOPA–induced

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dyskinesia in Parkinson's disease (Ferré et al., 2010). The D1R–D3R heteromers display

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higher affinity for DA compared to D1R alone, which results in an increase of G protein signaling and cAMP accumulation. Functional selectivity/biased agonism may be also related

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to the formation of homo- and heteromers. In this respect, it has been shown that

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heterodimerization of D3R with D1R abolishes agonist-induced D1R internalization induced by D1R agonists while enables internalization of the D1R/D3R complex in response to the

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paired D1R and D3R stimulation, a β-arrestin-dependent mechanism (Fiorentini et al., 2008). Most of the evidence supporting the formation of GPCR dimers and oligomers comes from

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heterologous systems, therefore the existence of such signaling complexes in the native

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context as well as their biological significance have been questioned (Lambert et al., 2014;

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Frederick et al., 2015), being extremely difficult to dissociate downstream crosstalk from the actual physical interaction of two receptors in a signaling complex (Frederick et al., 2016; Han et al., 2009; Urizar et al., 2011).

Pharmacological, genetic and human post-mortem studies have demonstrated the central role of D3R in the pathophysiology and treatment of schizophrenia, drug addiction, PD and depressive disorders, as discussed below. Addiction still represents the main research field with potential clinical application for D3R ligands. However, recent data have underscored the potential use of D3R ligands in diseases not previously taken into account, such as glaucoma. A number of D3R selective compounds has been developed and further helped the study of D3R location and function. A patent survey published in 2013 reports more than 110 submitted patent applications concerning D3R selective ligands (Sokoloff et al., 2013); however none of them has yet reached clinical approval, mainly because they did not fulfill

ACCEPTED MANUSCRIPT requirements of pharmacokinetics and/or safety. On the other hand, thanks to current experimental tools, some approved drugs, used since long time and believed to act through

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D2R binding, have been reviewed and are now considered to act, at least in part, through

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D3R. Here, we first review the relevance of D3R in several patho-physiological conditions; thereafter, we discuss the determinants of D3R selectivity and the assessment of D3R

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occupancy in vivo through positron emission tomography (PET) studies; finally, we

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summarize the data supporting D3R-related clinical benefits for already market approved drugs. DA and DR exert several relevant functions also at the periphery, particularly at the

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level of the cardiovascular system and the kidney, involved in regulation of blood pressure, sodium balance, and renal and adrenal functions (for reviews see Missale et al., 1998; Choi et

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al., 2015; Zhang & Harris, 2015). A number of recent papers have investigated the D2R like

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receptors in the kidney in animal models. However, except DA itself, which can be used to

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treat shock, no drugs acting at D3R are yet approved with indications for cardiovascular or renal diseases. Therefore, the role of D3R at the periphery will not be further discussed here. We conclude that, based on currently available data, efforts in drug research and development are warranted to obtain novel selective D3R ligands endowed with adequate safety profile for clinical use in humans.

2.Diseases involving D3R transmission

2.1.Schizophrenia Schizophrenia is a disease affecting about 1% of population worldwide, characterized by abnormalities of behavior and thinking with inability to understand reality. The first-line pharmacological treatment for schizophrenia is represented by antipsychotics. Since long

ACCEPTED MANUSCRIPT time, antipsychotics were considered D2R antagonists (Kapur & Mamo, 2003), and later on reconsidered as D2R-like antagonist, to indicate their poorly selective binding at D2R, D3R

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and D4R. None of the antipsychotic currently available act as selective ligand for D3R

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(McCormick et al., 2010; Schotte et al., 1996); for example, in vivo human PET studies have shown that clozapine, olanzapine and risperidone poorly occupy D3R in the brain of patients

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with schizophrenia (Graff-Guerrero et al., 2009a; Mizrahi et al., 2011). In contrast with human

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studies, a number of D3R selective ligands have recently become available for animal studies, where they have been tested, together with genetic deletion, to sort out the role of

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D3R in schizophrenia. Available drug treatments are effective in improving positive symptoms (delusions, hallucinations), but show limited activity on negative symptoms (anhedonia, social

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withdrawal, lack of motivation) and on cognitive dysfunction. It has been suggested that

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blockade of D3R may impact cognitive impairment, but preclinical data are somehow

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conflicting; indeed, D3R-/- show a better performance than WT in a step-through passiveavoidance paradigm (Micale et al., 2010), while treatment with the D3R selective antagonist SB277011 does not improve the performance in the Morris water maze test (Tanyeri et al., 2015). On the other hand, while overexpression of D3R in striatum does not induce cognitive deficits, it disrupts motivation, suggesting that changes in D3R may be involved in the negative symptoms of schizophrenia (Simpson et al., 2014). Most antipsychotics, either first or second generation, do not display selectivity for D3R over D2R, but few compounds, including aripiprazole, blonanserin and cariprazine, show some D3R selectivity (Table 1). Asenapine has higher affinity at D3R compared to D2R, but displays higher affinity at some 5hydroxytryptamine (5-HT) receptor subtypes (Shahid et al., 2009). The therapeutic potential of antipsychotics in relation to their interaction with D3R is discussed below.

ACCEPTED MANUSCRIPT 2.2.Parkinson’s disease (PD) PD is a common degenerative disorder of the aging brain, affecting about 0.3% of the

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entire population and about 1% of the population older than 60. Clinically, PD is characterized

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by the triad of tremor at rest, slowness of voluntary movements and rigidity. The main biochemical abnormality in PD is the profound deficit in brain DA levels, primarily, but not

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exclusively, attributed to the loss in the substantia nigra of dopaminergic neurons projecting to

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the striatum. Current pharmacological treatment for PD aims at restoring dopaminergic transmission. The L-DOPA therapy remains the gold standard for the symptomatic treatment

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of PD (Poewe et al., 2010). The therapeutic response to L-DOPA typically consists of two components: the short-duration response, an improvement in motor disability lasting a few

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hours following the administration of a single dose; the long-duration response, a sustained

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benefit deriving from prolonged administration lasting many hours or days after

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discontinuation of treatment. Unfortunately, long-term L-DOPA treatment is associated with fluctuations in motor response and involuntary movements, known as L-DOPA induced dyskinesia (LID). LID occurs in nearly 80% of patients who have been on L-DOPA treatment for more than 5 years and also in patients with early onset (Sossi et al., 2006). D3R expression is decreased in the striatum of PD patients, but increases in patients exhibiting LID (Bezard et al., 2003; Guigoni et al., 2005). A number of animal studies supports a critical role of D3R in dyskinesia. Tardive dyskinesia induced by haloperidol in nonhuman primates correlates with upregulated D3R in striatum (Mahmoudi et al., 2014); moreover, lentiviralinduced D3R overexpression in the dorsal striatum leads to the appearance of dyskinetic behavior (Cote et al., 2014). On the other hand, genetic deletion of the D3R decreases LID without interfering with the antiparkinsonian effect of L-DOPA (Solis et al., 2015), while striatal injection of oligonucleotides antisense targeting D3R blocks the behavioral sensitization to the

ACCEPTED MANUSCRIPT effects of repeated L-DOPA treatment in hemiparkinsonian rats (van Kampen & Stoessl, 2003). Worthy of note, L-DOPA treatment increases D3R expression more in D1R- than in

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D2R-containing striatal neurons, supporting the notion that the D3R directly interacts with the

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D1R in the control of LID (Solis et al., 2015). A number of studies tried to modify LID by using D2R/D3R/D4R ligands. In some animal studies, D3R blockade seems effective in decreasing

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LID (Solis et al., 2015; Visanji et al., 2009), but others do not observe the same effect (Mela et

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al., 2010). This apparent discrepancy may be due to the selectivity of compounds used to target D3R; indeed, they bind to all D2R-like receptors (D2R, D3R and D4R). Furthermore,

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D3R may not be the sole DR subtype involved in LID, because L-745870, a compound

model of PD (Huot et al., 2012).

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reported as selective D4R antagonist, is able to decrease the severity of LID in a macaque

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D3R exhibits biased signaling and desensitization pattern in response to certain agonists,

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including DA (Gil-Mast et al., 2013; Westrich et al., 2010). Increased expression of D3R associated to selective desensitization may therefore contribute to the development of motor symptoms of PD and LID (Westrich et al., 2010). A recent study shows that SK609, a D3R agonist with atypical signaling properties, improves the performances of hemiparkinson rats and, when used as adjuvant with L-DOPA, reduces the motor symptoms induced by L-DOPA (Simms et al., 2016). Thus, available evidence indicates that D3R is implicated in LID and may represent a therapeutic target, but, to our knowledge, no clinical studies are planned or currently running to investigate whether or not LID may be counteracted by D3R selective ligands. Buspirone, an approved drug endowed with D3R antagonist activity (see also below) will be evaluated in a clinical trial for LID (NCT02589340). The potential therapeutic effect of buspirone in LID, however, is attributed to its 5-HT1A agonist activity, because it reduces the firing rate of the

ACCEPTED MANUSCRIPT neurons in the subthalamic nucleus of rats through 5-HT1A receptors (Sagarduy et al., 2016). Furthermore, in PD patients, the efficacy of buspirone in decreasing synaptic DA and the level of

serotonergic terminal function, as

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attenuating LID positively correlates with

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labeled with PET ligands for the serotonin transporter (Politis et al., 2014).

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2.3.Addiction

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The mesocorticolimbic DA system is largely responsible for the reward mechanisms which reinforce behaviors essential for survival, including food intake and sexual activity. However,

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drugs of abuse modify this system to sustain compulsive drug seek and administration (Ikemoto & Bonci, 2014), with loss of ability to control strong urges and continuous drug

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intake despite the awareness of its harmful consequences. The fifth edition of the Diagnostic

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and Statistical Manual of Mental Disorders combines the categories of “Substance Abuse”

(SUD).

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and “Substance Dependence” into a single disorder, termed “Substance Use Disorder”

Data obtained with highly selective D3R ligands in animal models show that D3R are involved not only in drug-related reward and drug-intake, but also in behavioral sensitization, including reinstatement and drug-seeking behavior (Heidbreder et al., 2005; Joyce & Millan, 2005). On the other hand, increases in D3R expression have been found in animals chronically exposed to psychostimulant drugs (Caine & Koob, 1993; Neisewander et al., 2004) as well as following chronic ethanol intake (Leggio et al., 2014). Consistent with preclinical studies, upregulation of D3R has been observed in post-mortem studies, in human brain chronically exposed to cocaine (Segal et al., 1997) and, more recently, in in vivo PET studies, in human brain chronically exposed to methamphetamine (Boileau et al., 2012). D3R antagonists counteract self-administration of diverse drugs of abuse under schedules where

ACCEPTED MANUSCRIPT the response requirements are high and inhibit relapse reinstatement (Heidbreder & Newman, 2010; Le Foll et al., 2005), which further point to a common mechanism for drug-induced

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compulsive behavior and suggest the potential of targeting D3R to treat SUD.

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In human studies few compounds endowed with D3R antagonist activity have been tested so far and none is currently approved for SUD. GSK598809 is a drug showing some

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promising effects in early clinical development. This compound was able to transiently reduce

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craving for cigarettes, with a dosage producing 72%–89% occupancy of D3R, as assessed by [11C]-(+)-PHNO in PET (Newman et al., 2012b). A study is currently running to analyze

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modulation of reward, impulsivity and emotional reactivity by GSK598809 (Paterson et al.,

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2.4.Anxiety/depression

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2015).

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Since longtime, alteration of dopaminergic neurotransmission has been postulated to be linked to depression. Initially, it was proposed that depression was associated to a decrease in extracellular concentration of DA (Braestrup et al., 1975), later on that antidepressants act, at least in part, by affecting extracellular DA (Dailly et al., 2004; Reneric & Lucki, 1998). The analysis of DA release and DR stimulation following antidepressant treatment is complicated by the fact that most antidepressants inhibit the reuptake of 5-HT, which further affects DA release in different brain areas including the mesolimbic D3R rich ones (Dremencov et al., 2009; Ichikawa & Meltzer, 1999; Parsons & Justice, 1993). As mentioned above, D3R expression is restricted to striatum and mesolimbic system. An impairment of the mesolimbic dopaminergic pathway might be responsible for anhedonia, one of the major symptoms of depression. A number of findings support this view; for example, D2R/D3R binding has been found to be higher in amygdala of depressed patients (Klimek et

ACCEPTED MANUSCRIPT al., 2002); on the other hand, long term treatment with antidepressant drugs modifies D3R mRNA expression in the shell of nucleus accumbens (Lammers et al., 2000). Behavioral data

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with D3R ligands in animal models are not very abundant; pramipexole, a non-ergoline D3R

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agonist reduces the immobility time in the forced swimming test when given for up to 14 days (Maj et al., 1997) and normalizes bulbectomy-induced hyperactivity in rats (Breuer et al.,

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2009); the more selective D3R ligand 7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT)

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exerts an antidepressant-like activity similar to pramipexole in the olfactory bulbectomized rat (Breuer et al., 2009). However, data obtained in D3R-/- mice suggest that D2R rather than

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D3R is responsible for the antidepressant-like activity observed for pramipexole in the mouse forced swim test, because it similarly reduces the immobility time in WT and D3R-/- (Siuciak &

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Fujiwara, 2004). Pramipexole seems to reduce anhedonia, an effect detected as increase in

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sucrose intake, when tested in the chronic mild stress model (Willner et al., 1994); however,

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in stressed animals, it increases sucrose intake above the control level, while it increases sucrose intake also in control unstressed animals (Willner et al., 1994). Thus, whether the antidepressant-like activity of pramipexole in animal models is related to D3R stimulation remains questionable.

Chronic stress is considered a key factor in the pathogenesis of depression. Activity of mesolimbic dopaminergic neurons in the reward circuit is a key determinant of behavioral susceptibility/resilience to chronic stress (Cao et al., 2010). D3R has been found to be downregulated following chronic stress, and this change is reversed by antidepressant treatments (Maj et al., 1998) or electroconvulsive therapy (Lammers et al., 2000), suggesting that enhanced dopaminergic neurotransmission mediated by D3R participates in adaptive changes to stress. However, two studies did not find depressive-like behavior, either in the basal level (locomotion, exploration) or in immobilization stress paradigms, in D3R -/-

ACCEPTED MANUSCRIPT (Chourbaji et al., 2008; Xing et al., 2013), though D3R-/- appear more resistant to stressful procedures than WT littermates (Leggio et al., 2008; Xing et al., 2013), show better

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performance in the elevated plus maze and are more sensitive to the anxiolytic effect of

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diazepam (Leggio et al., 2011; Leggio et al., 2015). Finally, extensive evidence indicates that depression is accompanied by downregulation of Brain-Derived Neurotrophic Factor (BDNF)

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and that antidepressant drug treatments reestablish, at least in part, BDNF levels (Bjorkholm

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& Monteggia, 2016). D3R expression is tightly controlled by BDNF (Guillin et al., 2001), such that changes in D3R expression and function may be mechanistically linked to BDNF in

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depression; whether or not this potential mechanism impacts on clinical outcomes remains to

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2.5.Glaucoma

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be determined.

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Ocular hypertension is the main risk factor for glaucoma, a progressive optic neuropathy, which is the leading cause of blindness in industrialized countries. The United Kingdom glaucoma treatment study concluded that the risk of progression of glaucoma is reduced by 19% per unit decrease in intraocular pressure (IOP), in both normotensive and hypertensive patients (Heijl, 2015). Increase of IOP is due to an imbalance between aqueous humor (AH) inflow and outflow. Production and drainage of AH take place at the processes of ciliary body and trabecular meshwork/uveoscleral pathway, respectively. Five classes of topical drugs are currently available in the market: beta-blockers, carbonic anhydrase inhibitors, prostaglandin derivatives, sympathomimetics and miotics. The first two classes are “inflow” drugs, the other three are “outflow” drugs (Bucolo et al., 2015). The idea that dopaminergic system may play a role in the regulation of AH hydrodynamics was first hypothesized in the eighties (Chiou, 1984; Virno et al., 1992). First, a group of DA antagonists was studied for their ability to

ACCEPTED MANUSCRIPT suppress the IOP recovery rate of rabbits infused with hypertonic saline (Chiou, 1984); thereafter, some ergoline derivatives endowed with D2R-like agonist activity were showed to

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decrease IOP in different animal models (Potter et al., 1984; Potter & Shumate, 1987). Few

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years later D1R and D2R agonists were tested in patients with high tension open angle glaucoma (Virno et al., 1992). This study showed that selective D1R agonists such as

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fenoldopam, topically administered as eye drops, induced a significant increase in IOP only in

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eyes with hydrodynamic disorders, and such hypertensive effect was inhibited only by the D1R selective antagonist SCH-23390 (Virno et al., 1992); on the other hand, the D2R agonist

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bromocriptine was able to decrease IOP (Virno et al., 1992). This latter result was confirmed by Elibol et al. (Elibol et al., 1992) demonstrating that bromocriptine 0.05% and 0.1% eye

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drops decreased the IOP in normal subjects by 22.05.8% and 28.49.8% from basal level, respectively. These pioneering studies opened the way to further investigations on the role of

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dopaminergic system in IOP modulation. The idea that AH production was stimulated by D1R and inhibited by D2R was emerging, and a new pharmacological approach was proposed using a simultaneous blockade of D1R and stimulation of D2R (Prunte et al., 1997). The potential role of D3R in lowering IOP was first proposed in 2000 (Chu et al., 2000), based on the observation that topical application of 7-OH-DPAT, a selective D3R agonist, was able to decrease IOP in rabbits, while a D3R selective antagonist, U99194A, reverted its effect (Chu et al., 2000). The suggested location of D3R in the eye is in sympathetic fibers afferent to ciliary body; thus, activation of D3R might block AH inflow. Our lab confirmed the role of D3R as potential pharmacological target for ocular hypotensive

drugs,

by means of

pharmacological approaches along with gene deletion study (Fig. 1, Bucolo et al., 2012; Platania et al., 2013). Since DRs have been identified in ciliary body of mice, it is likely that activation of D3R blocks AH production. From these fundamental studies we propose that the

ACCEPTED MANUSCRIPT ocular hypotensive effect elicited by classical D2R agonists such as bromocriptine, lergotrile, lisuride, pergolide, cianergoline and cabergoline in animals and humans (Ohia et al., 2005;

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Potter & Burke, 1982; Potter et al., 1998; Saha et al., 2001) is related to D3R rather than D2R

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stimulation (Bucolo et al., 2012; Platania et al., 2013), consistent with available data on their affinity at D2R and D3R (Table 2). Worthy of note, the above mentioned ergot derivatives are

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not selective for DR, they also bind 5-HT and adrenergic receptors. Taking account of that, it

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has been difficult, in the past years, to figure out the precise mechanism of these drugs in terms of IOP modulation until the use of D3R-/- mice and/or D3R selective ligands. In

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conclusion, dopaminergic system is pivotal to regulate IOP and D3R is the key DR subtype,

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representing a promising potential target for the treatment of glaucoma.

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3.Rational design of selective D3R ligands

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Development of selective D3R ligands has been so far challenging, due to the high sequence identity and homology shared by D2R and D3R. Medicinal and computational chemists have carried out several efforts in order to determine the molecular features of D3R selectivity. The resolution of crystallographic structure of D3R (Chien et al., 2010) is a milestone for the rational design of selective D3R ligands. Before this essential finding, drug discovery of D3R selective ligands mainly utilized the following approaches: i) extensive synthesis of congeneric series of ligands, starting from different chemical scaffolds, along with structure activity relationship (SAR) studies (Banala et al., 2011; Chen et al., 2008; Taylor et al., 2010); ii) ligand-based computational studies (i.e. quantitative SAR based on bioinformatics) including ligand alignments, multivariate statistical analysis of correlation of activity and physic-chemical features of ligands (Boeckler et al., 2005b; Cha et al., 2003; Nilsson et al., 1997; Zhang et al., 2012); iii) structure-based computational studies (i.e.

ACCEPTED MANUSCRIPT quantitative SAR based on prediction of 3D interaction of the ligand in the pocket of the modeled receptor), where D3R is modeled on other G protein-coupled receptors, mainly the

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β2 adrenergic receptor (Boeckler et al., 2005a; Carlsson et al., 2011; Liu et al., 2011; Lopez et

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al., 2010; Varady et al., 2003; Wang et al., 2010). The latter two approaches are often complementary to classical SAR studies. Few years after cloning D3R receptor, the

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aminotetraline derivative 7-OH-DPAT was identified as selective D3R agonist (Damsma et al.,

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1993; Levesque et al., 1992). The aminotetraline template was thereafter used also for design of antagonists, but it was soon abandoned due to its rapid in vivo clearance (Hackling &

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Stark, 2002). Later on, based on a tetrahydroisoquinoline scaffold, SB277011A was synthesized (Stemp et al., 2000) and characterized as a selective D3R antagonist (Reavill et

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al., 2000), but it was not further developed for clinical use, because it showed poor

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bioavailability in cynomolgus monkeys (Austin et al., 2001). Within the D2R-like agonists,

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pramipexole is an old drug, an aminothiazole designed from (+)-apomorphine, previously reported to act on presynaptic D2R, later identified as D3R (Schneider & Mierau, 1987). Pramipexole was identified as a preferential D3R agonist with negligible binding at 5-HT receptors (Mierau et al., 1995; Millan et al., 2002). Based on these findings, the chemical scaffold of pramipexole was further used for discovery of selective agonists or partial agonists (Chen et al., 2008; Ghosh et al., 2010; Zhen et al., 2016). Drug design of selective D3R ligands has led to development of bitopic D3R compounds (Murray et al., 1995; Murray et al., 1996). A bitopic ligand is characterized by two pharmacophores, the primary and the secondary one, joint through a linker. The mechanism for D3R preference of these bitopic compounds was elucidated in 2010, with the resolution of D3R structure (Chien et al., 2010). The crystal structure of D3R revealed a secondary binding pocket (allosteric pocket) next to the primary binding pocket (orthosteric pocket); in the secondary binding pocket, a secondary

ACCEPTED MANUSCRIPT pharmacophore interacts with residues of helix II-III and helix VII. Homology modeling and molecular dynamics of D3R and D2R, based on 3D structure of D3R, revealed that the

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secondary binding pockets of D3R and D2R have different conformations (Fig. 2). Occupancy

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of the secondary binding pocket by bitopic D3R selective compounds involves hydrophobic interaction with helix VII in particular with Tyr 365 of D3R, whereas interaction with D2R

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involves helices I-II-VII (Michino et al., 2013; Newman et al., 2012a; Platania et al., 2012).

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Furthermore, the tetramethylene linker seems to confer a favorable flexibility to bitopic compounds leading to higher D3R/D2R selectivity (Michino et al., 2013).

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Buspirone and its metabolites, 5-OH-buspirone and 6’-OH-buspirone, are bitopic compounds with a tetramethylene linker (Fig. 3). 5-OH-buspirone and 6’-OH-buspirone show

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D3R/D2R higher selectivity, though they have lower affinity at both D3R and D2R, compared to buspirone (Bergman et al., 2013). Prediction of binding energy (Gbinding) by docking

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calculations confirms that buspirone and its metabolites have higher affinity at D3R over D2R (Table 3); moreover, interaction of buspirone and its metabolites are in accordance to the bitopic interaction pattern described above (Fig. 3). Interestingly, while buspirone and its metabolites interact with the secondary extracellular loop of D3R through polar interactions, they establish mainly hydrophobic interactions at D2R (Fig. 3). Finally, the bitopic design strategy has produced some useful tools such as LS-3-48, a compound with D3R partial agonist activity, proposed as D3R selective radiotracer for PET studies (Rangel-Barajas et al., 2014) and GSK598809 (Micheli et al., 2010), a drug in clinical development for treatment SUD (Paterson et al., 2015).

4.In vivo imaging of D3R

ACCEPTED MANUSCRIPT In vivo imaging of DA receptors can be non-invasively obtained in PET studies, both in animals and humans, by using radiotracers which emit positrons and bind with adequate

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specificity and selectivity to DR subtypes. Since 1985 [11C]raclopride has been used to trace

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D2-like receptors (Farde et al., 1985). Raclopride, as well as other benzimide derivatives, binds with similar affinity to both D2R and D3R; its signal in vivo corresponds mainly to D2R,

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because in any brain area this subtype is more abundant than D3R. Later on, [11C]-(+)-propyl-

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hexahydro-naphtho-oxazin ([11C]-(+)-PHNO) was introduced in PET studies (Ginovart et al., 2006). PHNO is an agonist which again binds to both D2R and D3R, but, at variance with

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raclopride, shows as much as 30-50-fold selectivity for D3R, as demonstrated in vitro and in vivo by using D3R-/- and/or displacement with D3R selective antagonists (Narendran et al.,

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2006; Rabiner et al., 2009). However, the selectivity of PHNO is not sufficient to exclude D2R

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binding when used in PET studies; interpretation of PET data with [11C]-(+)-PHNO takes

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therefore advantage of the known anatomical distribution of DR subtypes, such that, in the substantia nigra and hypothalamus it is entirely attributed to D3R, in contrast with the dorsal striatum were it is attributed to D2R (being D2R much more abundant than D3R in this area). Furthermore, based on the known D3R expression, the signal can be somehow calibrated, considering substantia nigra as 100% and cerebellum as 0% (Tziortzi et al., 2011). In areas such as globus pallidus or ventral striatum, were the abundance of D2R and D3R is comparable, interpretation of [11C]-(+)-PHNO is more difficult (Payer et al., 2014). In recent years, PET with [11C]-(+)-PHNO has provided not only important insights on D3R expression in a number of pathological conditions, but also data on D3R occupancy by different drug treatments, providing some information on their affinity and selectivity. So far, in patients with schizophrenia-spectrum disorder (Graff-Guerrero et al., 2009b) as well as in individuals with clinical high risk for schizophrenia (Suridjan et al., 2013) no difference in [11C]-

ACCEPTED MANUSCRIPT (+)-PHNO binding has been found in caudate, putamen, ventral striatum, globus pallidus, substantia nigra and thalamus, compared to age- and sex-matched healthy controls. On the

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other hand, a number of studies have shed light on the receptor occupancy by antipsychotic

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drug treatments; in this respect, one study did not detect occupancy of D3R by risperidone and olanzapine (Mizrahi et al., 2011) while a more recent one reports observable occupancy

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in substantia nigra/ventral tegmental area, regions where [11C]-(+)-PHNO signal is entirely

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attributed to D3R (Girgis et al., 2015). The apparent discrepancy between the two studies may be related to the fact that D3R expression increases during chronic treatment (2.5 weeks

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in Mizrahi’s study vs acute bolus in Girgis’s study). A recent study shows in vivo evidence of D3R occupancy by the novel antipsychotic blonanserin through [11C]-(+)-PHNO PET signal in

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rat (Baba et al., 2015); the pharmacology of blonanserin will be further discussed below. A

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number of studies have investigated D3R expression in brain of subjects with SUD through

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[11C]-(+)-PHNO PET. In methamphetamine polydrug users [11C]-(+)-PHNO binding is higher in the substantia nigra and in the globus pallidus, and seems to be related to drug-use severity (Boileau et al., 2012). Similarly, [11C]-(+)-PHNO binding was found higher in the substantia nigra of cocaine addicted (Payer et al., 2014). In alcohol-dependent patients there are no changes in striatal [11C]-(+)-PHNO, but there is an increase in hypothalamus (Erritzoe, et al. 2014); the pathophysiological meaning of this finding is unclear. While [11C]-(+)-PHNO PET studies provide evidence of D3R upregulation in SUD, they have not indicated, so far, D3R changes associated to impulse control disorders. In PD patients with impulse control disorders following treatment with L-DOPA or DR agonists, there is no change in striatal [11C](+)-PHNO (Payer et al., 2015). Consistently, in pathological gambling, a form of behavioral addiction associated with elevated impulsivity, no significant differences have been found in the same brain areas (Boileau et al., 2013). This latter finding may also suggests that, unlike

ACCEPTED MANUSCRIPT what found in subjects with SUD, D3R upregulation is not implicated in reward and reinforcement mechanisms operating in pathological gambling. However, pathological

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gambling is associated with increased DA release in dorsal striatum, as measured by [11C]-

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(+)-PHNO displacement, which seems related to gambling severity (Boileau et al., 2014). In recent years, animal behavior paradigms recapitulating some features of human clinical tests

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(e.g. Iowa gambling task) have shown that D2R and D3R ligands do not affect decision

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making in rats, while D4R ligands show limited effects (Di Ciano et al., 2015). Interestingly, when win-associated audiovisual cues are added to the gambling task, D3R agonists

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increase the frequency of the disadvantageous choice, while

D3R antagonists have the

opposite effect (Barrus et al., 2016). These data suggest that D3R stimulation alters decision

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making associated to cued tasks, which may be relevant to both pathological gambling and

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SUD. Changes in DR expression may occur in PD, particularly following L-DOPA therapy,

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and contribute to LID. A recent study reported increased [11C]-(+)-PHNO binding in the globus pallidus of patients with LID. The findings support therapeutic strategies which target and diminish activity at D3R to prevent LID (Payer et al., 2016). Finally, we should mention that PET studies using [11C]-(+)-PHNO represent an invaluable tool to get relative estimate of DA release in D3R rich brain areas, following specific treatments and/or behavioral tasks or stimuli. However, these experiments do not directly deal with D3R recognition and will not be further discussed here.

5.Drug treatments targeting D3R

5.1.Aripiprazole

ACCEPTED MANUSCRIPT Aripiprazole (Abilify®) is a second generation antipsychotic acting as partial agonist at D2R/D3R and 5-HT1A (Burstein et al., 2005; Tadori et al., 2011). The partial agonist activity at

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D3R distinguishes aripiprazole from all other second-generation antipsychotics such as

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quetiapine, clozapine, olanzapine, ziprasidone, risperidone which act as competitive antagonists at D2R (Tadori et al., 2008). The partial agonist activity of aripiprazole and its

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selectivity at D3R has been confirmed by medicinal chemistry studies (Luedtke et al., 2012;

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Vangveravong et al., 2011). Furthermore, aripiprazole shows a slightly higher intrinsic activity in inhibiting forskolin-dependent adenylyl cyclase at D3R over the D2LR (Vangveravong et al.,

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2011). Recent data show that aripiprazole exerts antipsychotic properties similar to cariprazine, a new antipsychotic drug with potent D3R partial agonist activity, able to occupy

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in rodents D3R at dosages producing antipsychotic-like effects (Kiss et al., 2010; Watson et

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al., 2016). In particular, both cariprazine and aripiprazole cause dose-dependent reversal of

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delay-induced impairment in the novel object recognition (NOR) paradigm. Aripiprazole and cariprazine result equally active in reversing NOR deficits in rats given neonatal phencyclidine and housed in social isolation from weaning (Watson et al., 2016). Furthermore, both aripiprazole and cariprazine reverse subchronic phencyclidine-induced impairment in NOR in mice and rats (Nagai et al., 2009; Neill et al., 2016). However, unlike cariprazine, aripiprazole does not modify deficits in social behaviors in animals, corresponding to negative symptoms in schizophrenia (Watson et al., 2016). This data may reflect a difference in effectiveness of aripiprazole and cariprazine in treating negative symptoms, probably due to the different affinity at D3R of aripiprazole versus cariprazine. In vitro ligand binding and functional studies show, in fact, that cariprazine has a higher affinity at D3R as compared with aripiprazole (Kiss et al., 2010; Tadori et al., 2011).

ACCEPTED MANUSCRIPT Aripiprazole is one of the most effective antipsychotic drug as an add-on treatment in patients with depression resistant to drug treatment (Komossa et al., 2010). Several clinical

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studies show that aripiprazole added to standard antidepressant drugs results in significant

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and clinically meaningful improvement in treatment-resistant depressed patients (Berman et al., 2009; Berman et al., 2007; Marcus et al., 2008). These data led to the first approval by the

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US Food and Drug Administration (FDA) of a second-generation antipsychotic for adjunctive

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treatment to standard antidepressants in adults with major depressive disorder. The antidepressant activity of aripiprazole might by related to its peculiar partial agonist activity at

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D3R (Tadori et al., 2008). However, the pharmacological activity of aripiprazole could be more complex when considered in the context of DR heteromerization. In fact, certain actions of

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aripiprazole may reflect low efficacy stimulation of D2LR, D3R or D2LR/D3R receptors, while,

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other effects may reflect blockade of D3R and/or D2R/D3R heterodimers weakly coupled to

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transduction mechanisms. The observation that the partial agonist activity of aripiprazole at D2LR is abrogated upon co-expression of D3R (Novi et al., 2007) indicates that at D2LR/D3R heterodimers aripiprazole behaves as D2R antagonist. The receptor mechanism through which aripiprazole exerts its antidepressant action remains to be precisely defined, but its elucidation may provide a novel frame to reposition pharmacological targeting of D2R and D3R as an adjunct therapy for depressive disorders. Table S1 reports clinical trials currently running or recently completed regarding aripiprazole in different CNS diseases.

5.2.Blonanserin Blonanserin (Lonasen®) is approved in Japan and in Korea for the treatment of schizophrenia (a Phase III clinical trial using risperidone as comparator has been completed

ACCEPTED MANUSCRIPT in China, NCT01516424). Blonanserin has potent binding affinity at D2R (Ki = 0.28 nM), D3R (Ki = 0.28 nM) and 5-HT2A (Ki = 0.64 nM) with at least 33-fold selectivity for D2R/D3R

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receptors over other tested receptors (Baba et al., 2015). Clinically, blonanserin exhibits

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atypical antipsychotic properties, with efficacy against positive, negative and cognitive symptoms (Tenjin et al., 2013). Potential tolerability benefits of the drug in short-term trials

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include extrapyramidal symptoms lower than with haloperidol (Garcia et al., 2009) and a

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prolactin increase (hyperprolactinemia) milder than with risperidone (Takahashi et al., 2013). Of note, blonanserin in vitro displays a similar binding affinity at D2R and D3R (Baba et al.,

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2015; Tadori et al., 2011), but it extensively occupies rat D3R at dosages producing plasma levels equivalent to antipsychotic doses in human (Baba et al., 2015). Indeed, blonanserin

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administered in rats at its antipsychotic-like doses, displaces the binding of [3H]-(+)-PHNO,

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showing a higher percentage of occupancy of D3R than risperidone, olanzapine or

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aripiprazole (Baba et al., 2015). Thus, blonanserin, unlike most atypical antipsychotic drugs is also a potent D3R receptor antagonist. The preclinical evidence suggests that D3R influences cognition by modulating medial prefrontal cortex functions despite the relatively low density of D3R in this region (Nakajima et al., 2013). Microdialysis data indicate that D3R antagonists enhance DA and acetylcholine neurotransmission in prefrontal cortex of rodents (Barth et al., 2013). A recent report shows that blonanserin enhances cortical DA and acetylcholine neurotransmission in mice similarly to NGB2904, a recognized D3R antagonist (Huang et al., 2015). Such an effect on DA levels is consistent with a number of observations obtained in cognitive animal paradigms. Indeed, through D3R antagonism, blonanserin reverses the deficit in visual recognition memory induced by phencyclidine, an NMDA receptor antagonist, and augments dopaminergic neurotransmission in the medial prefrontal cortex (Hida et al., 2015). Furthermore, blonanserin potently reverses cognitive impairment induced by the D3R

ACCEPTED MANUSCRIPT agonist (+)-PD-128907 or by the NMDA receptor antagonist ketamine in common marmosets (Kotani et al., 2016). These preclinical data indicate that blonanserin targets D3R in the

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medial prefrontal cortex and improves cognitive performance in experimental model of

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schizophrenia. Thus, blonanserin may be useful in the management of the cognitive

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symptoms of schizophrenia, which still represents an unmet need.

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5.3.Buspirone

Buspirone (Buspar®), also available as generic, is approved by the FDA for the treatment

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of anxiety disorders and the short-term relief of anxiety. It is also available, with the same indication, in several European countries and Australia. Buspirone binds with comparable

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affinities, in the range 19-98 nM, to 5-HT1A receptors, where it behaves as partial agonist

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(Noel et al., 2014; Roth et al., 2000), and to D3R and D4R, where it behaves as antagonist

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(Bergman et al., 2013), whereas, unlike benzodiazepines, it does not significantly interact with GABAA receptors. Buspirone interacts also with D2R, but with an affinity 5-fold lower than for D3R; moreover, 5-hydroxybuspirone and 6’-hydroxybuspirone, two metabolites of buspirone, show a 7-20 fold higher selectivity for D3R over D2R (Bergman et al., 2013). The occupancy of DR subtypes by buspirone has been assessed in in vivo PET studies. In the primate brain, following intramuscular administration, buspirone displaces both [11C]-(+)-PHNO and [11C]raclopride binding to striatum, indicating binding to both D2R and D3R (Kim et al., 2014). However, following oral administration, buspirone displaces [11C]-(+)-PHNO binding in D3Rrich regions (globus pallidum and midbrain), but barely affects [11C]raclopride binding (Kim et al., 2014). The higher selectivity of oral buspirone towards D3R blockade is consistent with extensive

first

pass

metabolism

which

generates

5-hydroxybuspirone

and

6’-

hydroxybuspirone. These preclinical data have not been confirmed in humans, where a recent

ACCEPTED MANUSCRIPT study found that acute oral buspirone, even at relatively high doses (120 mg), produces only a modest (<25%) displacement of [11C]-(+)-PHNO binding in D3R-rich regions (Le Foll et al.,

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2016). Buspirone has proven effective in preclinical models of SUD (Heidbreder and

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Newman, 2010; Higley et al., 2011; Song et al., 2012). In rhesus monkeys, buspirone reduces cocaine, nicotine, and cocaine-nicotine self-administration (Bergman et al., 2013; Mello et al.,

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2013a; Mello et al., 2013b). Other studies in rodents show that buspirone inhibits also relapse or cocaine triggered by

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(reinstatement of drug seeking behavior) to methamphetamine

stress, cues, and drug priming (Newman et al., 2012b). We found that buspirone reduces

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ethanol intake in mice (Leggio et al., 2014) and that this effect is not related to 5-HT1A stimulation (Fig. 4). However, the recent human PET study mentioned above reported poor

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occupation of D3R with doses as high as 120 mg. Such a lack of D3R occupancy in vivo may

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reflect the lack of effects of buspirone on ethanol intake found in several small trials (Fawcett

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et al., 2000; Malec et al., 1996). Buspirone has shown some efficacy in other SUDs, but its action is often referred to its anxiolytic effect (which is chiefly attributed to 5-HT1A receptor binding). Furthermore, samples are often too small and/or available data conflicting to draw positive conclusions for buspirone in cannabis SUD (McRae-Clark et al., 2009; Marshall et al., 2014; McRae-Clark et al., 2015), in heroin SUD (Rose et al., 2003), in cocaine SUD (Giannini et al., 1993; Winhusen et al., 2014). Currently, no clinical studies for alcoholism are running, but several studies are examining the effect of buspirone in other SUDs (Table S2). Of note, a clinical trial is planned to start soon, to evaluate the efficacy of buspirone in combination with amantadine on LID (NCT02589340). The therapeutic potential of buspirone in this condition has been related to 5HT1A receptor stimulation, but might also take advantage of the antagonism at D3R, given

ACCEPTED MANUSCRIPT that dyskinesia induced by L-DOPA or DR agonists is characterized by D3R upregulation in striatum (Solis et al., 2015).

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Table S2 reports clinical trials currently running or recently completed regarding buspirone

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in different CNS diseases.

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5.4.Cabergoline

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Cabergoline (Caberlin®, Dostinex® and Cabaser®) is an ergot derivative approved for hyperprolactinemia and PD; it is also used in a number of endocrine disorders and it has been

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tested in SUDs, such as cocaine dependence (Leiderman et al., 2005). Cabergoline is a potent agonist at D2R and D3R subtypes, and it also possesses significant affinity at 5-HT

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receptors such as 5-HT1A and 5-HT2A–B–C (Millan et al., 2002; Newman-Tancredi et al., 2002).

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Docking of cabergoline into the binding pocket of D3R resulted in a best scored pose similar

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to those reported for other D3R agonists (Kortagere et al., 2011; Platania et al., 2012); cabergoline’s pose involved the salt bridge between the protonated nitrogen N6 of the tretracyclic ergoline ring system and Asp 110. Cabergoline has been shown to significantly decrease IOP in different species (Platania et al., 2013; Sharif et al., 2009). In particular, we demonstrated that topical application of cabergoline, significantly decreased, in a dosedependent manner, the intraocular pressure in WT, both in an ocular normotensive group ( -9, -5 and -2 mmHg with 5%, 1%, and 0.1%, respectively) and an ocular hypertensive group (Fig. 1), with a prolonged effect in this latter group. On the contrary, in D3R-/-, either normotensive or with ocular hypertension, cabergoline treatment did not affect IOP (Platania et al., 2013) showing the key role of D3R in the mechanism of action of this drug. These data suggest that cabergoline may be useful in the management of IOP dysfunction, however clinical translational studies are warranted.

ACCEPTED MANUSCRIPT

5.5.Cariprazine

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Cariprazine (Vraylar®) is an antipsychotic approved by FDA for schizophrenia and bipolar

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disorder and currently submitted for approval to European Medicines Agency (EMA). It acts as a D2R and D3R partial agonist, with high selectivity towards the D3R; the reported Ki

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values are 0.5 nM for D2R and 0.1 nM for D3R (Agai-Csongor et al., 2012; Kiss et al., 2010)

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thus, at least theoretically, cariprazine should act as an antagonist at DR in presence of high dopamine levels, whereas would stimulate DR when in the presence of low dopamine level.

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Cariprazine is also a 5-HT1A receptor partial agonist, though with an affinity considerably lower than at DR. In vivo PET studies show 49% D2R/D3R occupancy in the striatum and

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30% 5-HT1A receptor occupancy in the raphe following 300 μg/kg cariprazine (Seneca et al.,

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2011). Many antipsychotics act on D2R located in dorsal striatum, whereas cariprazine acts

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more on D3R, mainly located in the ventral striatum; this selectivity of cariprazine for D3R may account for the reduced extrapyramidal side effects compared to other antipsychotics . In preclinical animal models cariprazine displays ‘antipsychotic-like’ activity, including the conditioned avoidance response and inhibition of amphetamine-induced hypermotility, without inducing ‘cataleptic-like’ activity (Gyertyan et al., 2011). Furthermore, cariprazine improves scopolamine-induced deficits in a rat water-labyrinth task (Gyertyan et al., 2011) and phencyclidine-induced deficits in rat and mouse tests of visual recognition memory, attentional set shifting/reversal learning and social interaction/social recognition memory (Zimnisky et al., 2013). More recently, cariprazine has been shown to revers impairment in both NOR and in social interaction induced in rat by phencyclidine (Neill et al., 2016; Watson et al., 2016); worthy of note, these effects occurred following a dose lower than those reducing locomotor activity (Neill et al., 2016). Cariprazine also attenuates the decrease in

ACCEPTED MANUSCRIPT sucrose intake in rats subjected to chronic mild stress, an animal paradigm of depression (Papp et al., 2014); the reduced sucrose intake is considered a model of anhedonia, a

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negative symptom associated to schizophrenia and depression. These data, showing that

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cariprazine improves cognition and reduces anhedonia, suggest a potential efficacy of cariprazine in schizophrenia negative symptoms (Papp et al., 2014). Unlike other atypical

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antipsychotics, cariprazine shows a high and balanced occupancy of both D2R and D3R in

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animals as well as in humans (Kiss et al., 2010). Clinical data are consistent with animal data, indicating a significant improvement of both positive and negative symptoms with cariprazine

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versus placebo (Durgam et al., 2015; Durgam et al., 2014; Kane et al., 2015); insomnia, extrapyramidal symptoms, sedation and akathisia are the most common reported adverse

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events. Interestingly, clinical improvements were reported at low doses (Durgam et al.,

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2016c), at which D2R are supposedly less occupied than D3R, because of cariprazine’s

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selectivity for D3R. Despite the evidence available from animal data, pro-cognitive effects of cariprazine in schizophrenic patients have not yet clearly demonstrated. As mentioned above, cariprazine has been shown to attenuate the chronic mild stressinduced decrease in sucrose intake in rats which has been suggested by some groups to be a model of anhedonia a symptom associated to depression (Papp et al.,2014). Cariprazine has been studied in major depressive disorder, as an adjunctive therapy in inadequate responders to antidepressants. The recently published results of a randomized, double-blind, placebocontrolled study suggests that adjunctive cariprazine 2-4.5 mg/d is effective and generally well tolerated in adults with major depressive disorder who had inadequate responses to standard antidepressants (Durgam et al., 2016a). Positive results have been also obtained from phase II and III clinical trials for bipolar mania (Calabrese et al., 2015; Durgam et al., 2016b; Sachs

ACCEPTED MANUSCRIPT et al., 2015) leading to approval by the FDA of cariprazine also for the treatment of manic or mixed episodes associated with bipolar disorder (McCormack, 2015).

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Table S3 reports clinical trials currently running or recently completed regarding

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cariprazine in different CNS diseases.

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6.Concluding Remarks

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In this paper we reviewed available evidence indicating that D3R is critically involved in a number of disorders, including schizophrenia, PD, SUD, anxiety, depression, glaucoma, and

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for this reason D3R represents a therapeutic target for pharmacotherapy. In recent years, PET imaging with [11C]-(+)-PHNO allowed researchers to assess in vivo the occupancy of

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D3R by a given drug treatment and/or their down- or up-regulation which may be a

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consequence of the disease as well as the consequence of exposure to a D3R ligand used in

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therapy. However, due to the high sequence identity and homology shared by D2R and D3R, pharmaceutical research and development has not lead so far to the approval of any selective D3R ligand to be used in human therapy. Finding useful molecular determinants of D3R selectivity seems presently achievable, thanks to the resolution of crystallographic structure of D3R in 2010, which serves as template for molecular modeling studies. Few drugs endowed with significant D3R affinity are available in the market. Some of them, such as buspirone and cabergoline, since longtime, others, such as aripiprazole, blonanserin and cariprazine, have been approved more recently. These drugs display peculiar therapeutic effects which may be, at least in part, related to their interaction with D3R. Worthy of note, these drugs have been evaluated in many clinical trials and still are under evaluation to assess their efficacy to treat conditions like schizophrenia, particularly the cognitive symptoms, SUD, LID, depressive disorders resistant to classical antidepressant therapy, etc. (see also Tables S1, S2 and S3).

ACCEPTED MANUSCRIPT Since these drugs are already available and do show a relevant interaction with D3R, they may provide new information on the significance of targeting D3R to cure the disorders

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mentioned above. On the other hand, they may also be considered as chemical scaffolds to

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develop novel, more selective D3R ligands, which will provide additional therapeutic value.

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Conflict of interest statement

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The authors declare that there are no conflicts of interest.

Acknowledgment

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PON 01_01434 _REACT.

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This work was supported by Finanziamento Ricerca di Ateneo – FIR Unict 7E646B and

ACCEPTED MANUSCRIPT References

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5HT1A

5HT2A

5HT2B

5HT2C

D1R D2LR D2SR D3R

Asenapine

8.6 [a]

10.15 9.75 [a] [a]

10.46 8.85 [a] [a]

8.9 [a]

Aripiprazole

8.57 [a]

8.02 [a]

9.59 [a]

7.55 [a]

6.09 [a]

8.94 [a]

Ziprasidone

9.01 [a]

9.51 [a]

9.08 [a]

9.01 [a]

8.45 [a]

Quietapine

6.78 [a]

6.81 [a]

7.33 [a]

5.98 [a]

Olanzapine

5.82 [a]

8.88 [a]

8.41 [a]

8.41 [a]

Risperidone

6.75 [a]

9.69 [a]

7.99 [a]

Clozapine

7.06 [a]

8.39 [a]

Haloperidol

6.29 [a]

Amisulpride

Sulpiride

PT

pKi

NU

Table 1. Binding of antipsychotics to serotonergic and dopaminergic receptors. D4R D5R

8.95 ND [a]

8.91 [a]

8.85 [a]

6.89 5.77 [b] [a]

8.09 [a]

7.99 [a]

8.35 [a]

7.33 6.81 [b] [a]

6.71 [a]

6.38 [a]

6.32 [a]

6.41 [a]

5.85 7.80 [b] [a]

7.93 [a]

7.67 [a]

7.58 [a]

7.46 [a]

7.75 7.04 [b] [a]

8.17 [a]

7.68 [a]

8.21 [a]

8.07 [a]

8.16 [a]

8.21 7.80 [b] [a]

8.79 [a]

8.56 [a]

7.64 [a]

6.87 [a]

6.81 [a]

6.66 [a]

7.33 6.63 [b] [a]

7.28 [a]

6.48 [a]

5.79 [a]

8.2 [a]

8.84 [a]

8.76 [a]

8.56 [a]

8.83 6.90 [b] [a]

5.8 [b]

5.08 [b]

7.88 [b]

5 [b]

5 [b]

8.89 [b]

ND

8.62 [b]

5.63 5 [b] [b]

5 [b]

ND

ND

ND

5 [b]

8 [b]

ND

8.07 [b]

7.26 ND [b]

Remoxipride ND

5.31 [b]

ND

ND

ND

7.21 [c]

ND

6.73 [c]

6 [c]

Cariprazine

8.59 [d]

7.73 [d]

9.24 [d]

6.87 [d]

ND

9.31 [d]

9.16 [d]

10.07 ND [d]

Trazodone

6.93 [b]

7.35 [b]

7.11 [b]

6.65 [b]

5.42 [b]

5.38 [b]

ND

ND

6.15 5 [b] [b]

Blonaserin

6.09

9.09

ND

7.57

5.97

9.84

ND

9.3

6.82 5.58 [e]

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TE

D

MA

9.38 [a]

SC

RI

8.84 [a]

ND

ND

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Buspirone

[e]

[e]

7.54 [b]

6.86 [b]

6.67 [b]

[e]

[e]

[e]

6.31 [b]

ND

7.2 [f]

ND

[e]

[e]

7.52 [f]

6.97 ND [b]

PT

ND, not determined; a, Shahid et al.,2009; b, Roth et al., 2000; c, Burstein et al., 2005; d, Kiss et al., 2010; e, Tenjin et al., 2013; f, Leggio et al., 2014.

DR subtype

Aqueous hydrodynamics

Bromocriptine

D2R/D3R

outflow [a]

Pergolide

D2R/D3R

Lergotrile

Species

D3R Ki(nM)

D2R Ki(nM)



H

29.4 [j]

38.6 [j]

outflow [b,c]



R, MK

1.08 [j]

16.4 [j]

D2R/D3R

outflow [d]



R, MK

ND

ND

Lisuride

D2R/D3R

outflow [b]



R, MK

0.95 [k]

0.79 [k]

Cianergoline

D2R/D3R

outflow [d]



R, MK

ND

ND

Cabergoline

D2R/D3R

outflow [e, f]



H, MK, M

0.43 [j]

0.74 [j]

Ibopamine Fenoldopam

MA

D

TE

AC CE P

7-OH-DPAT

IOP

NU

Compound

SC

RI

Table 2. Dopaminergic ligands and implications on aqueous humor hydrodynamics.

D3R

outflow [e,g,h]



R, MK, M

0.89 [k]

80.7 [k]

D1R

inflow [i]



H

ND

ND

D1R

inflow [i]



H

ND

ND

IOP, intraocular pressure; ND, not defined; H, human; R, rabbit; M, mouse; MK, monkey; a, Elibol et al., 1992; b, Potter et al., 1982; c, Siegel et al., 1987; d, Potter et al., 1987; e, Sharif et al., 2009; f. Platania et al., 2013; g, Chu et al., 2000; h. Bucolo et al., 2012; i. Virno et al.,1992; j. Tadori et al., 2011; k, Millan et al., 2002.

Table 3. Predicted binding free energies (G, Kcal/mol) and D2R/D3R energy ratios for buspirone, 5-OH-buspirone and 6’-OH-buspirone.

ACCEPTED MANUSCRIPT G D3R

G D2R

G D3R [a]

G D2R [a]

buspirone

-82.79

-63.10

-9.5

-8.57

5-OH-buspirone

-75.91

-69.88

-8.9

-7.31

6’-OH-buspirone

-75.87

-68.59

-8.2

-7.15

RI

SC

G G D2R/D3R D2R/D3R predicted experimental

PT

Compound

0.76

0.90

0.92

0.82

0.90

0.89

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D

MA

NU

a, Bergman et al., 2013 More negative energies correspond to better binding and docking scores. Structures of buspirone and its metabolites were retrieved from pubchem https://pubchem.ncbi.nlm.nih.gov/; whereas, structures of D3R and D2R were retrieved from Platania et al, 2012. Docking of compounds at D3R and D2R was carried out with Glide© Schrodinger, MM-GBSA calculations were carried out with Prime©, with energy minimization of residues far 10 Å from ligand. G from Bergman et al (2013) was calculated from G=RT ln Ki, R= 1.985 E-3 kcal/mol; K= 298.15. G D2R/D3R are reported instead of Ki D3R/D2R; those rations are equivalent to Ki D3R/D2R; because more negative energy corresponds to lower Ki and ratios are reported as absolute values.

ACCEPTED MANUSCRIPT Legends to figures Fig. 1. Effect of the D3R agonist 7-OH-DPAT (A) and cabergoline (B) on intraocular pressure

PT

(IOP) in WT and D3R-/- mice with steroid-induced ocular hypertension. 7-OH-DPAT (1%)

RI

and cabergoline (1%) ocular formulations were instilled in the mouse eye. Modified from

SC

Bucolo et al. (2012) and Platania et al. (2013).

NU

Fig. 2. Evolution of binding pockets of D3R and D2R after molecular dynamics. Pockets

MA

generated by Fpocket server are represented as colored clusters of spheres. Left panels represent D3R (green ribbons) and right panels represent D2 LR (cyan ribbons), before (A, B)

D

and after (C, D) molecular dynamics. The red circles target the orthosteric binding pocket

AC CE P

From Platania et al., 2012.

TE

whereas the black circles highlight the secondary binding pocket of D3R.

Fig. 3. Buspirone is a bitopic preferential D3R antagonist. Panel A. Schematic representation of binding of a bitopic compound (buspirone) at D3R receptor. Panel B. 2D interaction diagram of buspirone docked into D3R binding pocket. Panel C. Interaction diagram of buspirone docked into D2R binding pocket.

Fig. 4. Inhibition of ethanol intake by buspirone is related to D3R antagonism in mice. A) Effect of vehicle (VEH), Buspirone (BUSP, 3 mg/kg, intraperitoneally) and the selective 5HT1A agonist, 8-OH-DPAT (1 mg/kg, intraperitoneally) in WT exposed to the Drinking in the Dark paradigm. Buspirone, but not 8-OH-DPAT, significantly decreases ethanol intake. B) Hypothermia induced by 3 mg/kg buspirone or 1 mg/kg 8-OH-DPAT, an effect mediated by 5HT1A receptor stimulation. At the tested doses, 8-OH-DPAT, but not buspirone, induces a

ACCEPTED MANUSCRIPT long-lasting hypothermia. C) Buspirone significantly reduces ethanol intake in the Two Bottle Choice paradigm.

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Modified from Leggio et al., 2014.

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Figure 2

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Figure 4