Selective Dopamine D2 Receptor Antagonist Research Articles

D2 receptor-mediated miRNA-143 expression is associated with the effects of antipsychotic drugs on phencyclidine-induced schizophrenia-related locomotor hyperactivity and with Neuregulin-1 expression in mice

Non-coding RNA molecules such as miRNAs have emerged as critical regulators of neuronal functions. The present study investigates a role for miRNA-143, a highly conserved miRNA, in locomotorhyperactivity induced bythe psychotomimetic phencyclidine (PCP), a non-selective antagonist of the N-methyl-d-aspartate (NMDA) glutamate receptor. Following acute PCP administration to mice, the content of miRNA-143 was reduced in plasma, prefrontal cortex (PFC) and hippocampus, reaching a minimum after 2 h. The antipsychotics haloperidol and clozapine attenuated hyperlocomotion and the decrease in miR-143 expression induced by PCP, as did the selective D2 dopamine receptor antagonist eticlopride but not the selective D1 antagonist SCH23390. To further confirm D2 receptor-mediated miRNA-143 expression, HT-22 neuronal cell line and primary cortical cultured neuronswere studied. Stimulation of D2 receptors with the selective D2 receptor agonist quinpirole decreased expression of miRNA-143 in a time-dependent manner. This inhibition was blocked by pretreatment with eticlopride, indicating that the D2 receptor directly regulates the expression of miRNA-143. We further demonstrated that miRNA-143 directly targeted to the 3’ un-translated region of neuregulin-1 (NRG1) mRNA to reduce protein expression of NRG1 in HT-22 cells and that administration of the D2 receptor agonist quinpirole to mice enhanced expression of NRG1 in PFC. The present data provide the first evidence that D2 receptors are involved in the expression ofmiRNA-143 in association with antipsychotic drug action and the developmental regulator NRG1.

The highly selective dopamine D3R antagonist, R-VK4-40 attenuates oxycodone reward and augments analgesia in rodents

Prescription opioid abuse is a global crisis. New treatment strategies for pain and opioid use disorders are urgently required. We evaluated the effects of R-VK4-40, a highly selective dopamine (DA) D3 receptor (D3R) antagonist, on the rewarding and analgesic effects of oxycodone, the most commonly abused prescription opioid, in rats and mice. Systemic administration of R-VK4-40 dose-dependently inhibited oxycodone self-administration and shifted oxycodone dose-response curves downward in rats. Pretreatment with R-VK4-40 also dose-dependently lowered break-points for oxycodone under a progressive-ratio schedule. To determine whether a DA-dependent mechanism underlies the impact of D3 antagonism in reducing opioid reward, we used optogenetic approaches to examine intracranial self-stimulation (ICSS) maintained by optical activation of ventral tegmental area (VTA) DA neurons in DAT-Cre mice. Photoactivation of VTA DA in non-drug treated mice produced robust ICSS behavior. Lower doses of oxycodone enhanced, while higher doses inhibited, optical ICSS. Pretreatment with R-VK4-40 blocked oxycodone-enhanced brain-stimulation reward. By itself, R-VK4-40 produced a modest dose-dependent reduction in optical ICSS. Pretreatment with R-VK4-40 did not compromise the antinociceptive effects of oxycodone in rats, and R-VK4-40 alone produced mild antinociceptive effects without altering open-field locomotion or rotarod locomotor performance. Together, these findings suggest R-VK4-40 may permit a lower dose of prescription opioids for pain management, potentially mitigating tolerance and dependence, while diminishing reward potency. Hence, development of R-VK4-40 as a therapy for the treatment of opioid use disorders and/or pain is currently underway.This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.

Aberrant insular cortex connectivity in abstinent alcohol-dependent rats is reversed by dopamine D3 receptor blockade.

A few studies have reported aberrant functional connectivity in alcoholic patients, but the specific neural circuits involved remain unknown. Moreover, it is unclear whether these alterations can be reversed upon treatment. Here, we used functional MRI to study resting state connectivity in rats following chronic intermittent exposure to ethanol. Further, we evaluated the effects of SB‐277011‐a, a selective dopamine D3 receptor antagonist, known to decrease ethanol consumption. Alcohol‐dependent and control rats (N = 13/14 per group), 3 weeks into abstinence, were administered SB‐277011‐a or vehicle before fMRI sessions. Resting state connectivity networks were extracted by independent component analysis. A dual‐regression analysis was performed using independent component maps as spatial regressors, and the effects of alcohol history and treatment on connectivity were assessed. A history of alcohol dependence caused widespread reduction of the internal coherence of components. Weaker correlation was also found between the insula cortex (IC) and cingulate cortices, key constituents of the salience network. Similarly, reduced connectivity was observed between a component comprising the anterior insular cortex, together with the caudate putamen (CPu‐AntIns), and the posterior part of the IC. On the other hand, postdependent rats showed strengthened connectivity between salience and reward networks. In particular, higher connectivity was observed between insula and nucleus accumbens, between the ventral tegmental area and the cingulate cortex and between the VTA and CPu‐AntIns. Interestingly, aberrant connectivity in postdependent rats was partially restored by acute administration of SB‐277011‐a, which, conversely, had no significant effects in naïve rats.

Selective D2 and D3 receptor antagonists oppositely modulate cocaine responses in mice via distinct postsynaptic mechanisms in nucleus accumbens.

The dopamine D3 receptor (D3R) has emerged as a promising pharmacotherapeutic target for the treatment of several diseases including schizophrenia, Parkinson's disease, and substance use disorders. However, studies investigating the D3R's precise role in dopamine neurotransmission or how it may be exploited to modulate responses to drugs of abuse have produced contrasting results, in part because most D3R-targeted compounds often also interact with D2 receptors (D2R). To resolve this issue, we set out to systematically characterize and compare the consequences of selective D2R or D3R antagonists on the behavioral-stimulant properties of cocaine in mice, and to identify putative neurobiological mechanisms underlying their behavior-modifying effects. Pretreatment with the selective D2R antagonist L-741,626 attenuated, while pretreatment with the selective D3R antagonist PG01037 enhanced, the locomotor-activating effects of both acute cocaine administration as well as sensitization following repeated cocaine dosing. While both antagonists potentiated cocaine-induced increases in presynaptic dopamine release, we report for the first time that D3R blockade uniquely facilitated dopamine-mediated excitation of D1-expressing medium spiny neurons in the nucleus accumbens. Collectively, our results demonstrate that selective D3R antagonism potentiates the behavioral-stimulant effects of cocaine in mice, an effect that is in direct opposition to that produced by selective D2R antagonism or nonselective D2-like receptor antagonists, and is likely mediated by facilitating D1-mediated excitation in the nucleus accumbens. These findings provide novel insights into the neuropharmacological actions of D3R antagonists on mesolimbic dopamine neurotransmission and their potential utility as pharmacotherapeutics.

Dopamine D3R antagonist VK4-116 attenuates oxycodone self-administration and reinstatement without compromising its antinociceptive effects.

Prescription opioids such as oxycodone are highly effective analgesics for clinical pain management, but their misuse and abuse have led to the current opioid epidemic in the United States. In order to ameliorate this public health crisis, the development of effective pharmacotherapies for the prevention and treatment of opioid abuse and addiction is essential and urgently required. In this study, we evaluated-in laboratory rats-the potential utility of VK4-116, a novel and highly selective dopamine D3 receptor (D3R) antagonist, for the prevention and treatment of prescription opioid use disorders. Pretreatment with VK4-116 (5-25 mg/kg, i.p.) dose-dependently inhibited the acquisition and maintenance of oxycodone self-administration. VK4-116 also lowered the break-point (BP) for oxycodone self-administration under a progressive-ratio schedule of reinforcement, shifted the oxycodone dose-response curve downward, and inhibited oxycodone extinction responding and reinstatement of oxycodone-seeking behavior. In addition, VK4-116 pretreatment dose-dependently enhanced the antinociceptive effects of oxycodone and reduced naloxone-precipitated conditioned place aversion in rats chronically treated with oxycodone. In contrast, VK4-116 had little effect on oral sucrose self-administration. Taken together, these findings indicate a central role for D3Rs in opioid reward and support further development of VK4-116 as an effective agent for mitigating the development of opioid addiction, reducing the severity of withdrawal and preventing relapse.

Methcathinone and 3-Fluoromethcathinone Stimulate Spontaneous Horizontal Locomotor Activity in Mice and Elevate Extracellular Dopamine and Serotonin Levels in the Mouse Striatum

Methcathinone (MC) and 3-fluoromethcathinone (3-FMC) are well-known members of the synthetic cathinone derivatives, the second most abused group of novel psychoactive substances (NPS). They are considered as methamphetamine-like cathinones, as they elicit their psychostimulatory effects via inhibition of monoamine uptake and enhanced release. The present study examines the effects of MC and 3-FMC on the spontaneous locomotor activity of mice and extracellular levels of dopamine and serotonin in the mouse striatum. Both MC and 3-FMC produced a dose-dependent increase of horizontal locomotor activity, but no significant changes in rearing behavior were observed. The locomotor stimulation induced by MC and 3-FMC is mediated by activation of dopaminergic neurotransmission, as selective D1-dopamine receptor antagonist, SCH 23390, abolished the effects of both drugs. In line with pharmacological data obtained by previous in vitro studies, MC and 3-FMC produced potent increases of extracellular dopamine and serotonin levels in the mouse striatum. Taken together, results presented within this study confirm previous findings and expand our knowledge on the pharmacology of MC and 3-FMC along with their behavioral effects.

Discovery of CNS-Like D3R-Selective Antagonists Using 3D Pharmacophore Guided Virtual Screening.

The dopamine D3 receptor is an important CNS target for the treatment of a variety of neurological diseases. Selective dopamine D3 receptor antagonists modulate the improvement of psychostimulant addiction and relapse. In this study, five and six featured pharmacophore models of D3R antagonists were generated and evaluated with the post-hoc score combining two survival scores of active and inactive. Among the Top 10 models, APRRR215 and AHPRRR104 were chosen based on the coefficient of determination (APRRR215: R2training = 0.80; AHPRRR104: R2training = 0.82) and predictability (APRRR215: Q2test = 0.73, R2predictive = 0.82; AHPRRR104: Q2test = 0.86, R2predictive = 0.74) of their 3D-quantitative structure–activity relationship models. Pharmacophore-based virtual screening of a large compound library from eMolecules (>3 million compounds) using two optimal models expedited the search process by a 100-fold speed increase compared to the docking-based screening (HTVS scoring function in Glide) and identified a series of hit compounds having promising novel scaffolds. After the screening, docking scores, as an adjuvant predictor, were added to two fitness scores (from the pharmacophore models) and predicted Ki (from PLSs of the QSAR models) to improve accuracy. Final selection of the most promising hit compounds were also evaluated for CNS-like properties as well as expected D3R antagonism.

Selective dopamine D3 receptor antagonist YQA14 inhibits morphine-induced behavioral sensitization in wild type, but not in dopamine D3 receptor knockout mice.

Increasing preclinical evidence demonstrates that dopamine D3 receptor (D3R) antagonists are a potential option for the treatment of drug addiction. The reinstatement of the addiction can be triggered by environmental stimuli that acquire motivational salience through repeated associations with the drug's effects. YQA14 is a novel D3R antagonist that has exhibited pharmacotherapeutic efficacy in reducing cocaine and amphetamine reward and relapse to drug seeking in mice. In this study we investigated the effects of YQA14 on morphine-induced context-specific locomotor sensitization in mice. We showed that repeated injection of YQA14 (6.25-25 mg/kg every day ip) prior to morphine (10 mg/kg every day sc) not only inhibited the acquisition, but also significantly attenuated the expression of morphine-induced locomotor sensitization. Furthermore, in the expression phase, one single injection of YQA14 (6.25-25 mg/kg, ip) dose-dependently inhibited the expression of morphine-induced behavioral sensitization. Moreover, YQA14 inhibited the expression of morphine-induced behavioral sensitization in wild mice (WT), but not in D3R knockout (D3R-/-) mice in the expression phase. In addition, D3R-/- mice also displayed the reduction in the expression phase compared with WT mice. In summary, this study demonstrates that blockade or knockout of the D3R inhibits morphine-induced behavior sensitization, suggesting that D3R plays an important role in the pathogenesis and etiology of morphine addiction, and it might be a potential target for clinical management of opioid addiction.

Selective dopamine D3 receptor antagonism significantly attenuates stress-induced immobility in a rat model of post-traumatic stress disorder.

Post-traumatic stress disorder (PTSD) is a debilitating psychiatric syndrome that occurs in individuals exposed to extremely threatening or traumatic events. In both animals and humans, dopamine (DA) function appears to be dysregulated in brain areas involved in the conditioned fear response(s) that underlie PTSD. In this study, we determined the effect of the selective DA D3 receptor antagonists YQA14A (6.25, 12.5 and 25 mg/kg i.p.) and SB-277011A (6 mg/kg i.p.) on tone-induced fear (assessed by measuring freeze time) in a modified version of the single-prolonged stress (SPS) model of PTSD in adult male Sprague-Dawley rats. Rats pretreated with vehicle and then subjected to restraint stress, forced swim and random foot shock (SPS) in the presence of a distinctive tone, displayed a significantly increased tone-induced contextual freeze time and fecal pellet mass following re-exposure to the tone. Rats pretreated with a single i.p. injection of 6.25 or 12.5 mg/kg of YQA14 or 6 mg/kg of SB-277011A showed significantly attenuated contextual freeze time in the presence of the tone when tested 14 days after exposure to SPS. Overall, our results indicate that selectively antagonizing DA D3 receptors significantly decreases freezing time caused by an environment previously associated with stress. If our findings can be extrapolated to humans with PTSD, they suggest that DA D3 receptors may play a role in the pathophysiology of PTSD, and may have therapeutic utility for the clinical management of PTSD.

The basolateral amygdala dopaminergic system contributes to the improving effect of nicotine on stress-induced memory impairment in rats

Stress seems to be an important risk factor in the beginning and continuing stages of cigarette tobacco smoking in humans. Considering that both of nicotine administration and stress exposure affect cognitive functions including memory formation, the aim of the present study was 1) to evaluate the effect of subcutaneous (s.c.) administration of nicotine on memory formation under stress and 2) to assess the possible role of the basolateral amygdala (BLA) dopamine D1 and D2 receptors in the effect of nicotine on stress-induced memory retrieval impairment. Adult male wistar rats were bilaterally implanted in the BLA. A step-through type passive avoidance task was used to measure memory retrieval. To induce acute stress, the animals were placed on an elevated platform. The results showed that pre-test exposure to 20 and 30 min stress, but not 10 min, impaired memory retrieval. Nicotine administration (0.05 mg/kg, s.c.) improved stress-induced memory retrieval impairment. The activation of the BLA dopamine receptors via bilateral microinjection of apomorphine (0.025–0.4 μg/rat), a non-selective dopamine receptor agonist, potentiated the effect of nicotine on stress-induced memory retrieval impairment. Interestingly, intra-BLA microinjection of SCH23390 (a selective dopamine D1 receptor antagonist; 0.02–0.5 μg/rat) or sulpiride (a selective dopamine D2 receptor antagonist; 0.02–0.5 μg/rat) dose-dependently inhibited nicotine-induced improvement of the stress amnesic effect. Taken together, it can be concluded that stress-induced impairment of memory retrieval can be improved by nicotine administration. Moreover, the dopaminergic neurotransmission in the BLA through D1 and D2 receptors mediates the improving effect of nicotine on stress-induced memory retrieval impairment.

Identification and Characterization of ML321: A Novel and Selective D2 Dopamine Receptor Antagonist with Predicted Atypical Antipsychotic Properties

Dopamine receptors (DARs) belong to the G protein‐coupled receptor (GPCR) superfamily, whose members play critical roles in cellular signaling, especially modulating the transfer of information within the nervous system. Amongst DARs, the D2 dopamine receptor (D2R) is one of the most validated drug targets in neuropsychiatry and there is a strong correlation between the clinical doses of antipsychotics and their potencies for blocking D2Rs. However, current FDA‐approved antipsychotic drugs typically cross‐react with other GPCRs, leading to many serious off‐target side‐effects. A potent and highly selective D2R antagonist would thus be extremely valuable for the treatment of psychosis and related CNS disorders. Using high throughput screening techniques, we have identified, optimized, and characterized a lead D2R antagonist with high selectivity – ML321. In a functional profiling screen of 168 different GPCRs, ML321 showed relatively little reactivity beyond inhibition of the D2R, and to a lesser extent the D3R, demonstrating exceptional GPCR selectivity. Interestingly, we found that the binding of ML321 to the D2R is entirely dependent on Na+. PET imaging studies in non‐human primates demonstrated that ML321 can penetrate the CNS and occupy the D2R in a dose‐dependent manner. Preliminary in vitro ADME studies showed that ML321 is metabolically stable and did not block hERG channels, whereas pharmacokinetic studies in mice demonstrated elevated plasma and brain levels of ML321 for up to 8 hrs. Behavioral paradigms in rats demonstrated that that ML321 can selectivity antagonize a D2R‐mediated response (hypothermia) while not affecting a D3R‐mediated response (yawning) using the same dose of drug, thus demonstrating good in vivo selectivity. We also investigated the effects of ML321 in animal models that are predictive of antipsychotic efficacy in humans. We found that ML321 can attenuate both PCP and amphetamine‐induced locomotor activity and pre‐pulse inhibition (PPI) in a dose‐dependent manner. Importantly, using doses that were maximally effective in the locomotor and PPI studies, ML321 promoted little‐no catalepsy compared with the nonselective antipsychotic haloperidol. This latter observation suggests that ML321 may produce fewer extrapyramidal (motor) side‐effects, a common problem with FDA‐approved antipsychotics. Overall, these results suggest that the ML321 scaffold could serve as a lead compound for the development of an improved therapeutic with greatly reduced side‐effects for treating schizophrenia and other psychotic syndromes.Support or Funding InformationNINDS IRPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

From Bench to Bedside: Translating the Prolactin/Vasoinhibin Axis.

The prolactin/vasoinhibin axis defines an endocrine system, in which prolactin (PRL) and vasoinhibins regulate blood vessel growth and function, the secretion of other hormones, inflammatory and immune processes, coagulation, and behavior. The core element of the PRL/vasoinhibin axis is the generation of vasoinhibins, which consists in the proteolytic cleavage of their precursor molecule PRL. Vasoinhibins can interact with multiple different partners to mediate their effects in various tissues and anatomical compartments, indicating their pleiotropic nature. Based on accumulating knowledge about the PRL/vasoinhibin axis, two clinical trials were initiated, in which vasoinhibin levels are the target of therapeutic interventions. One trial investigates the effect of levosulpiride, a selective dopamine D2-receptor antagonist, on retinal alterations in patients with diabetic macular edema and retinopathy. The rationale of this trial is that the levosulpiride-induced hyperprolactinemia resulting in increased retinal vasoinhibins could lead to beneficiary outcomes in terms of a vasoinhibin-mediated antagonization of diabetes-induced retinal alterations. Another trial investigated the effect of bromocriptine, a dopamine D2-receptor agonist, for the treatment of peripartum cardiomyopathy. The rationale of treatment with bromocriptine is the inhibition of vasoinhibin generation by substrate depletion to prevent detrimental effects on the myocardial microvascularization. The trial demonstrated that bromocriptine treatment was associated with a high rate of left ventricular recovery and low morbidity and mortality. Therapeutic interventions into the PRL/vasoinhibin axis bear the risk of side effects in the areas of blood coagulation, blood pressure, and alterations of the mental state.

The effects of local microinjection of selective dopamine D1 and D2 receptor agonists and antagonists into the dorsal raphe nucleus on sleep and wakefulness in the rat

The effects of the dopamine (DA) D1 and D2 receptor agonists SKF38393, bromocriptine and quinpirole, respectively, on spontaneous sleep were analyzed in adult rats prepared for chronic sleep recordings. Local administration of the DAergic agonists into the dorsal raphe nucleus (DRN) during the light phase of the light-dark cycle induced a significant reduction of rapid-eye movement sleep (REMS) and the number of REM periods. Additionally, bromocriptine and quinpirole significantly increased wakefulness (W). Opposite, the microinjection into the DRN of the DA D1 and D2 receptor antagonists SCH23390 and sulpiride, respectively, significantly augmented REMS and the number of REM periods. Pretreatment with SCH23390 and sulpiride prevented the effects of SKF38393 and bromocriptine, respectively, on sleep variables. Our results tend to indicate that DAergic neurons located in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) contribute to the regulation of predominantly W and REMS by DRN serotonergic neurons.

Separating the agony from ecstasy: R(–)-3,4-methylenedioxymethamphetamine has prosocial and therapeutic-like effects without signs of neurotoxicity in mice

S,R(+/–)-3,4-methylenedioxymethamphetamine (SR-MDMA) is an amphetamine derivative with prosocial and putative therapeutic effects. Ongoing clinical trials are investigating it as a treatment for post-traumatic stress disorder (PTSD) and other conditions. However, its potential for adverse effects such as hyperthermia and neurotoxicity may limit its clinical viability. We investigated the hypothesis that one of the two enantiomers of SR-MDMA, R-MDMA, would retain the prosocial and therapeutic effects but with fewer adverse effects. Using male Swiss Webster and C57BL/6 mice, the prosocial effects of R-MDMA were measured using a social interaction test, and the therapeutic-like effects were assessed using a Pavlovian fear conditioning and extinction paradigm relevant to PTSD. Locomotor activity and body temperature were tracked after administration, and neurotoxicity was evaluated post-mortem. R-MDMA significantly increased murine social interaction and facilitated extinction of conditioned freezing. Yet, unlike racemic MDMA, it did not increase locomotor activity, produce signs of neurotoxicity, or increase body temperature. A key pharmacological difference between R-MDMA and racemic MDMA is that R-MDMA has much lower potency as a dopamine releaser. Pretreatment with a selective dopamine D1 receptor antagonist prevented SR-MDMA-induced hyperthermia, suggesting that differential dopamine signaling may explain some of the observed differences between the treatments. Together, these results indicate that the prosocial and therapeutic effects of SR-MDMA may be separable from the stimulant, thermogenic, and potential neurotoxic effects. To what extent these findings translate to humans will require further investigation, but these data suggest that R-MDMA could be a more viable therapeutic option for the treatment of PTSD and other disorders for which SR-MDMA is currently being investigated.

Ameliorative effect of chlorpromazine hydrochloride on visceral hypersensitivity in rats: possible involvement of 5-HT2A receptor.

Visceral hypersensitivity is responsible for pathogenesis of irritable bowel syndrome (IBS). Therefore, its prevention can help avoid abdominal pain and discomfort in IBS. To find candidate drugs for visceral hypersensitivity, we screened existing medicines for their ability to prevent visceral sensitivity induced by colorectal distension (CRD) in rats and identified chlorpromazine, a typical antipsychotic drug, as a candidate compound. In this study, we investigated the effect of chlorpromazine on visceral hypersensitivity. Visceral sensitivity (visceromotor response) was monitored by measuring the electrical activity of the abdominal external oblique muscle contraction in response to CRD using a barostat apparatus. Visceral hypersensitivity was induced by a colonic instillation of sodium butyrate or acetic acid in neonates. Oral administration of chlorpromazine suppressed butyrate-induced visceral hypersensitivity to CRD. Interestingly, atypical antipsychotic drugs, quetiapine and risperidone, ameliorated butyrate-induced visceral hypersensitivity, whereas the typical antipsychotic drugs, haloperidol and sulpiride, did not. Pharmacological analysis using specific inhibitors showed that a selective 5-HT2A receptor antagonist, ketanserin, suppressed butyrate-induced visceral hypersensitivity, whereas a selective dopamine D2 receptor antagonist, L-741626, did not. Furthermore, the 5-HT2A receptor agonist AL-34662 stimulated visceral sensitivity to CRD in healthy control rats but not in butyrate-treated rats. These findings suggest that increased 5-HT levels in the colon contribute to the induction of visceral hypersensitivity. Our results indicate that chlorpromazine ameliorates visceral hypersensitivity and that the 5-HT2A receptor is a potential therapeutic target for treating abdominal pain and discomfort in IBS.

Abstract 1191: The dopamine (DA) D3 receptor antagonists (PG01037, NGB2904, SB-277011A, and U99194) significantly attenuate ABCG2-mediated multidrug resistance

Abstract ATP-binding family G2 (ABCG2) transporters are known to produce multidrug resistance (MDR) and limit successful cancer chemotherapy. Using molecular modeling, we obtained data indicating that highly potent and selective dopamine (DA) D3 receptor antagonists had significant docking scores for the active site of the ABCG2 transporter. In this in vitro study, we determined the effect of the D3 receptor antagonists (PG01037, NGB2904, SB-277011A, and U99194) on MDR resulting from the overexpression of ABCG2 transporters. The D3 receptor antagonists alone did not significantly affect the viability of HEK293/ABCG2, H460/MX-20, S1-M1-80 or A549-MX-10 cells, which overexpress ABCG2 transporters. However, the D3 receptor antagonists (PG01037, NGB2904, SB-277011A, and U99194) significantly increased the efficacy of the anticancer drugs mitoxantrone and doxorubicin in the above mentioned cell lines. Efflux studies indicated that both PG01037 and NGB2904 significantly decreased the efflux of rhodamine 123 from H460-MX20 cells. Interestingly, PG01037 and NGB2904 significantly decreased the expression levels of the ABCG2 protein levels as shown by immunocytochemical and Western blot analysis. This suggests that D3 antagonists inhibit both function and expression of ABCG2 transporters at relevant non-toxic concentrations. In conclusion, our in vitro results indicate that PG01037, NGB2904, SB-277011A, and U99194 reverse resistance to mitoxantrone and doxorubicin mediated by overexpression of ABCG2 transporters. Further mechanistic and animal studies are warranted to establish the clinical use of D3 receptor antagonists in reversing ABCG2-mediated MDR. Citation Format: Noor Hussein, Haneen Amawi, Charles R. Ashby, Karthikeyan Chandrabose, Roopali Mittal, Ryann Christman, Piyush Trivedi, Amit Tiwari. The dopamine (DA) D3 receptor antagonists (PG01037, NGB2904, SB-277011A, and U99194) significantly attenuate ABCG2-mediated multidrug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1191. doi:10.1158/1538-7445.AM2017-1191

Potent haloperidol derivatives covalently binding to the dopamine D2 receptor

The dopamine D2 receptor (D2R) is a common drug target for the treatment of a variety of neurological disorders including schizophrenia. Structure based design of subtype selective D2R antagonists requires high resolution crystal structures of the receptor and pharmacological tools promoting a better understanding of the protein-ligand interactions. Recently, we reported the development of a chemically activated dopamine derivative (FAUC150) designed to covalently bind the L94C mutant of the dopamine D2 receptor. Using FAUC150 as a template, we elaborated the design and synthesis of irreversible analogs of the potent antipsychotic drug haloperidol forming covalent D2R-ligand complexes. The disulfide- and Michael acceptor-functionalized compounds showed significant receptor affinity and an irreversible binding profile in radioligand depletion experiments.

Larval settlement and metamorphosis of the invasive biofouler, Mytilopsis sallei, in response to ions and neuroactive compounds

ABSTRACTIn this study, we investigated larval settlement and metamorphosis of the invasive fouler Mytilopsis sallei exposed to ions, neurotransmitters and blockers inhibiting their respective actions. Excess K+ effectively induced larval settlement and metamorphosis, while the voltage-gated potassium channel blocker, TEA, significantly inhibited the K+ inducing effect, suggesting that a voltage-gated potassium channel may play a role in M. sallei settlement and metamorphosis. Excess Ca2+ did not induce larval settlement and metamorphosis, while Mg2+ and inhibited both. Among the neurotransmitters, GABA did not induce M. sallei larvae to settle and metamorphose at 10−6–10−4 M concentrations, while 5 × 10−5–10−4 M l-DOPA (a dopamine precursor), 5 × 10−6–10−4 M dopamine (an epinephrine precursor) and 5 × 10−5–10−4 M epinephrine significantly induced larval settlement and metamorphosis, indicating the presence of an epinephrine biosynthesis pathway in this species and its role in the regulation of larval settlement and metamorphosis. Furthermore, the inducing effect of dopamine on M. sallei settlement and metamorphosis was inhibited by SCH23390, a selective D1 dopamine receptor antagonist. Similarly, the inducing effect of epinephrine was inhibited by chlorpromazine, an α1-adrenergic antagonist, suggesting that the D1 dopamine receptor and α1-adrenoceptor may play active roles in the processes of settlement and metamorphosis of M. sallei larvae. Here, we have shown for the first time the responses of larval settlement and metamorphosis of dreissenid mussels to pharmacologically active compounds. The results provide new insights into the biochemical mechanisms underlying larval settlement and metamorphosis of M. sallei, which may be useful to develop effective strategies to control this invasive fouling organism.

Optimization of a Novel D2 Dopamine Receptor Antagonist Scaffold Reveals Exceptionally Selective and Potent Lead Molecules

FDA‐approved antipsychotics are primarily D2 dopamine receptor (D2R) antagonists, however they generally lack receptor selectivity and interact with other GPCRs, including D3Rs. Despite its clear clinical importance, there are few compounds that selectively modulate the D2R. This is due to structural similarities in the orthosteric binding sites of the D2‐like receptors (D2R, D3R, and D4R), as well as other biogenic amine receptors. To find better therapeutic candidates, as well as probe compounds to investigate D2R pathophysiology, we sought to identify highly selective D2R antagonists by implementing a high‐throughput screening (HTS) campaign using more than 400,000 unique compounds in the Molecular Libraries Screening Center Network library. Most of the hit compounds that were identified as antagonists of the D2R were also antagonists of the closely related D3R. However, several compounds were found to exhibit high affinity for the D2R with low affinity for the D3R. One such compound (MLS6916) served as our lead scaffold for optimization. MLS6916 is ~700 fold selective for the D2R vs. D3R as determined in functional β‐arrestin recruitment assays and radioligand binding competition assays. Importantly, MLS6916 was also examined for functional activity at 168 different GPCRs and showed no agonist activity on any GPCR tested, and only exhibited antagonist activity at the D2‐like receptors. Preliminary investigations, however, revealed that this initial hit compound was metabolically unstable. To optimize this hit scaffold, we conducted an iterative medicinal chemistry campaign with the goals of increasing selectivity, potency, and engendering metabolic stability. Over 70 analogs of the MLS6916 parent compound were synthesized to dissect the influence of various constituents of the molecule and to construct structure‐activity relationships. Analogs were analyzed for activity on D2R, D3R, and D4R using both radioligand binding and functional assays to determine affinities and potencies for the D2‐like receptors. In addition, analogs were tested for solubility, permeability, and stability. Several lead compounds were identified with Ki values of < 50 nM for the D2R with > 1,000‐fold selectivity vs. the D3R. These selectivity trends were confirmed in functional β‐arrestin recruitment assays. Importantly, lead compounds with enhanced metabolic stability and permeability were identified. Further analysis and optimization of these lead compounds will provide new insight into highly selective D2R antagonists for the treatment and understanding of dopamine‐related disorders.Support or Funding InformationNINDS Intramural Research Program

P.1.l.019 - Influence of personality traits on tinnitus severity perception, anxiety and depressive symptoms

Electrical and chemical stimulation of the dorsal periaqueductal gray (dPAG), deep layers of the superior colliculus (dlSC), and inferior colliculus (IC) causes freezing and escape behavior in rodents. Systemic injections of the selective dopamine D2 receptor antagonist sulpiride increased the number of switch-off responses (SORs) to light and auditory evoked potentials in response to loud sounds. Dopamine D2 receptor inhibition in the IC was shown to enhance unconditioned fear. Nevertheless, the role of dopamine receptors in the dlSC and dPAG in the mediation of unconditioned fear has not yet been demonstrated.The purpose of the present study was to characterize the effects of sulpiride injections (4 and 8 μg/0.2 μl) in the dlSC and dPAG in rats that were subjected to unconditioned fear paradigms.Switch-off responses to light and exploratory behavior in the elevated plus maze were used to evaluate unconditioned fear in rats.Intra-dlSC microinjections of sulpiride increased the number of SORs to light. Intra-dlSC and intra-dPAG injections of sulpiride reduced the number of entries into and time spent on the open arms and decreased end-arm exploration and head dipping in the elevated plus maze.These findings suggest that dopamine, through D2 receptors in the dlSC and dPAG, is involved in defense reactions that are organized in the midbrain tectum.