In addition to the serotonin 5-HT2A receptor (5-HT2AR), the dopamine D2 receptor (D2R) is a key therapeutic target of antipsychotics for the treatment of schizophrenia. The inactive state structures of D2R have been described in complex with the inverse agonists risperidone (D2Rris) and haloperidol (D2Rhal). Here we describe the structure of human D2R in complex with spiperone (D2Rspi). In D2Rspi, the conformation of the extracellular loop (ECL) 2, which composes the ligand-binding pocket, was substantially different from those in D2Rris and D2Rhal, demonstrating that ECL2 in D2R is highly dynamic. Moreover, D2Rspi exhibited an extended binding pocket to accommodate spiperone's phenyl ring, which probably contributes to the selectivity of spiperone to D2R and 5-HT2AR. Together with D2Rris and D2Rhal, the structural information of D2Rspi should be of value for designing novel antipsychotics with improved safety and efficacy.

Concentration of hyaluronic acid (HA) in the lungs increases in idiopathic pulmonary fibrosis (IPF). HA is involved in the organization of fibrin, fibronectin, and collagen. HA has been proposed to be a biomarker of fibrosis and a potential target for antifibrotic therapy. Hyaluronidase (HD) breaks down HA into fragments, but is a subject of rapid hydrolysis. A conjugate of poloxamer hyaluronidase (pHD) was prepared using protein immobilization with ionizing radiation. In a model of bleomycin-induced pulmonary fibrosis, pHD decreased the level of tissue IL-1β and TGF-β, prevented the infiltration of the lung parenchyma by CD16+ cells, and reduced perivascular and peribronchial inflammation. Simultaneously, a decrease in the concentrations of HA, hydroxyproline, collagen 1, total soluble collagen, and the area of connective tissue in the lungs was observed. The effects of pHD were significantly stronger compared to native HD which can be attributed to the higher stability of pHD. Additional spiperone administration increased the anti-inflammatory and antifibrotic effects of pHD and accelerated the regeneration of the damaged lung. The potentiating effects of spiperone can be explained by the disruption of the dopamine-induced mobilization and migration of fibroblast progenitor cells into the lungs and differentiation of lung mesenchymal stem cells (MSC) into cells of stromal lines. Thus, a combination of pHD and spiperone may represent a promising approach for the treatment of IPF and lung regeneration.

Wnt signaling affects fundamental development pathways by regulating cell proliferation and differentiation. Aberrant activation of Wnt/beta-catenin signaling promotes the development of several cancers and is an attractive target for chemopreventive and chemotherapeutic agents.

Endothelial dysfunction and destruction of the pulmonary microcirculation are important pathogenic factors in chronic obstructive pulmonary disease (COPD). In COPD, bronchial obstruction is associated with endothelial dysfunction. Thus, new pharmacological treatment options aimed at restoring the pulmonary endothelium represent a clinical need in COPD therapy. Notch1 has been shown to protect cells against apoptosis, inflammation, and oxidative stress caused by cigarette smoke extract (CSE). Therefore, drug which effect on Notch1 may be a potential therapeutic target for COPD in the future.

Approximately 50% of colorectal cancer (CRC) patients still die from recurrence and metastatic disease, highlighting the need for novel therapeutic strategies. Drug repurposing is attracting increasing attention because, compared to traditional de novo drug discovery processes, it may reduce drug development periods and costs. Epidemiological and preclinical evidence support the antitumor activity of antipsychotic drugs. Herein, we dissect the mechanism of action of the typical antipsychotic spiperone in CRC. Spiperone can reduce the clonogenic potential of stem-like CRC cells (CRC-SCs) and induce cell cycle arrest and apoptosis, in both differentiated and CRC-SCs, at clinically relevant concentrations whose toxicity is negligible for non-neoplastic cells. Analysis of intracellular Ca2+ kinetics upon spiperone treatment revealed a massive phospholipase C (PLC)-dependent endoplasmic reticulum (ER) Ca2+ release, resulting in ER Ca2+ homeostasis disruption. RNA sequencing revealed unfolded protein response (UPR) activation, ER stress, and induction of apoptosis, along with IRE1-dependent decay of mRNA (RIDD) activation. Lipidomic analysis showed a significant alteration of lipid profile and, in particular, of sphingolipids. Damage to the Golgi apparatus was also observed. Our data suggest that spiperone can represent an effective drug in the treatment of CRC, and that ER stress induction, along with lipid metabolism alteration, represents effective druggable pathways in CRC.

Coated vesicles prepared from bovine brain cerebral cortex exhibited [3H]5-hydroxytryptamine (5-HT, serotonin) and [3H]spiperone binding activities. The binding activities were localized in the inner core vesicles. Binding reached an equilibrium level by 30-45 min at 30 degreesC, and was reversed by the addition of 100 microM 5-HT for [3H]5-HT binding or 10 microM ketanserin for [3H]spiperone binding. The saturation binding experiments indicated a single class of binding sites for [3H]5-HT and [3H]spiperone with apparent Kd values of 2.4 and 1.75 nM, respectively. The binding of [3H]5-HT was displaced by 5-HT and 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT), but not by ketanserin. The binding of [3H]spiperone was displaced by spiperone and ketanserin but not by 5-HT or 8-OH-DPAT even at 1 mM. The coated vesicles were shown by immunoblotting assay to contain alpha-subunits of GTP-binding proteins, Galphas, Galphai2, Galphai3, Galphao and Galphaq/11. Forskolin-stimulated adenylate cyclase activity in the coated vesicles was inhibited to 80% of the control level by 5-HT or 8-OH-DPAT. These results suggested that 5-HT1A and 5-HT2A receptors are present in bovine brain coated vesicles and that the 5-HT1A receptors are coupled to adenylate cyclase activity via GTP binding proteins.

Some large antagonist ligands (ARC239, chlorpromazine, prazosin, spiperone, spiroxatrine) bind to the human α(2A) -adrenoceptor with 10- to 100-fold lower affinity than to the α(2B)- and α(2C)-adrenoceptor subtypes. Previous mutagenesis studies have not explained this subtype selectivity.

Ca(2+) activated Cl(-) channels (CaCC) are up-regulated in cystic fibrosis (CF) airway surface epithelia. The presence and functional properties of CaCC make it a possible therapeutic target to compensate for the deficiency of Cl(-) secretion in CF epithelia. CaCC is activated by an increase in cytosolic Ca(2+), which not only activates epithelial CaCCs, but also inhibits epithelial Na(+) hyperabsorption, which may also be beneficial in CF. Our previous study has shown that spiperone, a known antipsychotic drug, activates CaCCs and stimulates Cl(-) secretion in polarized human non-CF and CF airway epithelial cell monolayers in vitro, and in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) knockout mice in vivo. Spiperone activates CaCC not by acting in its well-known role as an antagonist of either 5-HT2 or D2 receptors, but through a protein tyrosine kinase-coupled phospholipase C-dependent pathway. Moreover, spiperone independently activates CFTR through a novel mechanism. Herein, we performed a mass spectrometry analysis and identified the signaling molecule that mediates the spiperone effect in activating chloride secretion through CaCC and CFTR. Proline-rich tyrosine kinase 2 (PYK2) is a non-receptor protein tyrosine kinase, which belongs to the focal adhesion kinase family. The inhibition of PYK2 notably reduced the ability of spiperone to increase intracellular Ca(2+) and Cl(-) secretion. In conclusion, we have identified the tyrosine kinase, PYK2, as the modulator, which plays a crucial role in the activation of CaCC and CFTR by spiperone. The identification of this novel role of PYK2 reveals a new signaling pathway in human airway epithelial cells.

Hyaluronidases are groups of enzymes that degrade hyaluronic acid (HA). To stop enzymatic hydrolysis we modified testicular hyaluronidase (HYAL) by activated polyethylene oxide with the help of electron-beam synthesis. As a result we received pegylated hyaluronidase (pegHYAL). Spiperone is a selective D2 dopamine receptor antagonist. It was demonstrated on the model of a single bleomycin damage of alveolar epithelium that during the inflammatory phase monotherapy by pegHYAL or spiperone reduced the populations of hematopoietic stem /progenitor cells in the lung parenchyma. PegHYAL also reduced the levels of transforming growth factor (TGF)-β, interleukin (IL)-1β, tumor necrosis factor (TNF)-α in the serum and lungs, while spiperone reduced the level of the serum IL-1β. Polytherapy by spiperone and pegHYAL caused the increase of the quantity of hematopoietic stem/ progenitor cells in the lungs. Such an influx of blood cell precursors was observed on the background of considerable fall level of TGF-β and the increase level of TNF-α in the serum and lungs. These results show pegHYAL reduced the bleomycin-induced fibrosis reaction (production and accumulation of collagen) in the lung parenchyma. This effect was observed at a single and repetitive bleomycin damage of alveolar epithelium, the antifibrotic activity of pegHYAL surpassing the activity of testicular HYAL. The antifibrotic effect of pegHYAL is enhanced by an additional instillation of spiperone. Therapy by pegHYAL causes the flow of CD31‒ CD34‒ CD45‒ CD44+ CD73+ CD90+ CD106+-cells into the fibrous lungs. These cells are incapable of differentiating into fibroblast cells. Spiperone instillation separately or together with pegHYAL reduced the MSC-like cells considerably. These data enable us to assume, that pegHYAL is a new and promising instrument both for preventive and therapy of toxic pneumofibrosis. The blockage of D2 dopamine receptors with the following change of hyaluronan matrix can be considered as a new strategy in treatment of pneumofibrosis.

Dopamine D₂ receptors (D₂R) are known to form transient homodimer complexes, of which the increased formation has already been associated with development of schizophrenia. Pharmacological targeting and modulation of the equilibrium of these receptor homodimers might lead to a better understanding of the critical role played by these complexes in physiological and pathological conditions. Whereas agonist addition has shown to prolong the D₂R dimer lifetime and increase the level of dimer formation, the possible influence of D₂R antagonists on dimerization has remained rather unexplored. Here, using a live-cell reporter assay based on the functional complementation of a split Nanoluciferase, a panel of six D₂R antagonists were screened for their ability to modulate the level of D2LR dimer formation. Incubation with the D₂R antagonist spiperone decreased the level of D2LR dimer formation significantly by 40-60% in real-time and after long-term (≥16 h) incubations. The fact that dimer formation of the well-studied A2a-D2LR dimer was not altered following incubation with spiperone supports the specificity of this observation. Other D₂R antagonists, such as clozapine, risperidone, and droperidol did not significantly evoke this dissociation event. Furthermore, molecular modeling reveals that spiperone presents specific Tyr1995.48 and Phe3906.52 conformations, compared to clozapine, which may determine D₂R homodimerization.

The dopamine D3 receptor (D3R) is a molecular target for both first-generation and several recently-developed antipsychotic agents. Following stable expression of this mEGFP-tagged receptor, Spatial Intensity Distribution Analysis indicated that a substantial proportion of the receptor was present within dimeric/oligomeric complexes and that increased expression levels of the receptor favored a greater dimer to monomer ratio. Addition of the antipsychotics, spiperone or haloperidol, resulted in re-organization of D3R quaternary structure to promote monomerization. This action was dependent on ligand concentration and reversed upon drug washout. By contrast, a number of other antagonists with high affinity at the D3R, did not alter the dimer/monomer ratio. Molecular dynamics simulations following docking of each of the ligands into a model of the D3R derived from the available atomic level structure, and comparisons to the receptor in the absence of ligand, were undertaken. They showed that, in contrast to the other antagonists, spiperone and haloperidol respectively increased the atomic distance between reference α carbon atoms of transmembrane domains IV and V and I and II, both of which provide key interfaces for D3R dimerization. These results offer a molecular explanation for the distinctive ability of spiperone and haloperidol to disrupt D3R dimerization.

Acute exposure to kainic acid (KA) induces neurochemical changes in dopaminergic systems in the brain and the aim of the present study was to investigate the acute toxicity of KA upon dopamine D2 receptors. Adult rats were injected intraperitoneally with either saline or 16 mg/kg KA. Brains were removed after 4 h. Membrane homogenates were prepared from seven brain regions and in addition, frozen coronal sections were sectioned for comparative quantitative autoradiographic analysis. Dopamine D2 receptors were characterised by saturation studies using [125I]iodosulpiride, [3H]raclopride and [3H]spiperone. KA produced a 2-fold decrease in receptor affinity for [125I]iodosulpiride and a 2-fold increase in receptor density in all regions studied except striatum. Quantitative autoradiography with [125I]iodosulpiride showed similar increases in D2 labelling following KA except in caudate putamen, nucleus accumbens and olfactory tubercle. In contrast, there was no change in [3H]spiperone binding in whole brain minus striatum nor in striatum alone after KA treatment. KA produced a significant increase in Bmax for [3H]raclopride in whole brain minus striatum and in striatum alone with minimal changes in affinity. These findings demonstrate acute changes in rat brain dopamine D2 receptors labelled with [125I]iodosulpiride and [3H]raclopride but not [3H]spiperone after KA treatment predominantly in extra striatal regions.

Background and Purpose: Drug repositioning is a promising strategy for discovering new therapeutic strategies for cancer therapy. We investigated psychotropic drugs for their antitumor activity because of several epidemiological studies reporting lower cancer incidence in individuals receiving long term drug treatment. Experimental Approach: We investigated 27 psychotropic drugs for their cytotoxic activity in colorectal carcinoma, glioblastoma and breast cancer cell lines. Consistent with the cationic amphiphilic structure of the most cytotoxic compounds, we investigated their effect on mitochondrial and lysosomal compartments. Results: Penfluridol, ebastine, pimozide and fluoxetine, fluspirilene and nefazodone showed significant cytotoxicity, in the low micromolar range, in all cell lines tested. In MCF7 cells these drugs caused mitochondrial membrane depolarization, increased the acidic vesicular compartments and induced phospholipidosis. Both penfluridol and spiperone induced AMPK activation and autophagy. Neither caspase nor autophagy inhibitors rescued cells from death induced by ebastine, fluoxetine, fluspirilene and nefazodone. Treatment with 3-methyladenine partially rescued cell death induced by pimozide and spiperone, whereas enhanced the cytotoxic activity of penfluridol. Conversely, inhibition of lysosomal cathepsins significantly reduced cell death induced by ebastin, penfluridol, pimozide, spiperone and mildly in fluoxetine treated cells. Lastly, Spiperone cytotoxicity was restricted to colorectal cancer and breast cancer and caused apoptotic cell death in MCF7 cells. Conclusions: The cytotoxicity of psychotropic drugs with cationic amphiphilic structures relied on simultaneous mitochondrial and lysosomal disruption and induction of cell death that not necessarily requires apoptosis. Since dual targeting of lysosomes and mitochondria constitutes a new promising therapeutic approach for cancer, particularly those in which the apoptotic machinery is defective, these data further support their clinical development for cancer therapy.

1. The release of glutamic acid and its modulation by 5-hydroxytryptamine (5-HT) in the human brain has been investigated in synaptosomal preparations from fresh neocortical samples obtained from patients undergoing neurosurgery to reach deeply sited tumours. 2. The Ca2+-dependent K+ (15 mM)-evoked overflow of glutamate was inhibited by 5-HT in a concentration-dependent manner (EC50 = 2.9 nM; maximal effect approximately 50%). The inhibition caused by 5-HT was antagonized by the 5-HT1/5-HT2 receptor antagonist methiothepin. The 5-HT1B/5-HT1D receptor agonist sumatriptan mimicked 5-HT (EC50 = 6.4 nM; maximal effect approximately 50%); the effect of sumatriptan was also methiothepin-sensitive. Selective 5-HT1A receptor antagonists could not prevent the inhibition of glutamate release by 5-HT. 3. The 5-HT1B/5-HT1D receptor ligand GR 127935 and the 5-HT2C/5-HT1B/5-HT1D receptor ligand metergoline were unable to prevent the 5-HT effect; instead they inhibited glutamate release, their effects being abolished by methiothepin. Some 5-HT1A receptor antagonists also displayed intrinsic agonist activity. 4. The effect of sumatriptan was prevented by ketanserin, a drug known to display much higher affinity for recombinant h 5-HT1D than for h 5-HT1B receptors. 5. We propose that neocortical glutamatergic nerve terminals in human brain cortex possess release-inhibiting presynaptic heteroreceptors that appear to belong to the h 5-HT1D subtype.

Background. Cerebral ischemia is known to produce brain damage and related behavioural deficits, including memory deficits and motor disorders. Evidence shows that EA significantly promotes recovery of neurological function and thus improves quality of life. Objective. Evidence exists for the involvement of catecholamines in human neuroplasticity. A better understanding of dopaminergic (DAergic) modulation in this process will be important. Methods. A total of 72 adult male Sprague-Dawley (SD) rats were divided into 6 groups: normal, model, EA, spiperone group, EA + spiperone group, and pergolide. The middle cerebral artery occlusion (MCAO) model was used in all 6 groups except the normal group. A behavioural assessment was conducted at 1, 3, 5, and 7 days after MCAO. The percent of brain infarct area was also determined 7 days after MCAO. Tyrosine hydroxylase (TH) and growth-associated protein 43 (GAP-43) fluorescence double labeling was performed in the striatum. Results. In this study, we found that EA at Fengchi (GB20) acupoints resulted in marked improvements based on a behavioural assessment. Both TTC staining and GAP-43 immunofluorescence labeling results showed that EA treatment reduced ischemia injury and promoted neuroplasticity compared with the model group. The D2R-selective agonist, pergolide, showed similar results, but these results were reversed by the D2R-selective antagonist, spiperone. We also found that there were more colocalization and expression of GAP-43 and TH in the EA and pergolide groups than those in the other groups. Conclusion. These results suggest that the neuroplasticity induced by EA was mediated by D2 autoreceptors in DAergic neurons.

Animal models have demonstrated a link between decreases in retinal dopamine levels and the development of form-deprivation myopia (FDM). However, the consistency of dopamine's role in the other major form of experimental myopia, that of lens-induced myopia (LIM), is less clear, raising the question as to what extent dopamine plays a role in human myopia. Therefore, to better define the role of dopamine in both forms of experimental myopia, we examined how consistent the protection afforded by dopamine and the dopamine agonist 6-amino-5,6,7,8-tetrahydronaphthalene-2,3-diol hydrobromide (ADTN) is between FDM and LIM.

Dopamine deficiency syndrome is known to cause cholinergic hyperactivity. Therefore, it was hypothesized that the said phenomenon may be due to enhanced cholinergic receptor functions. In the present study we examined the changes in striatal dopaminergic and cholinergic receptors in unilateral substantia nigra lesioned rats that showed vigorous ipsilateral rotation (total turns > 300) in response to apomorphine (1 mg kg(-1) ip). [3H] Spiperone ([3H]-SP) and [3H]-quinuclidinyl benzilate ([3H]-QNB) bindings were performed in the striata of the lesioned animals. There was no significant difference in the dissociation equilibrium constant values (Kd) between the lesioned and non-lesioned sides. However, a significant difference in the maximum receptor density (Bmax) of both [3H]-SP and [3H]-QNB bindings was observed between the lesioned and non-lesioned sides. The Bmax of [3H]-SP binding was significantly decreased on the lesioned side, whereas the Bmax of the [3H]-QNB binding was significantly increased. These results support the hypothesis that deficiencies of the dopaminergic system cause overactivity of the cholinergic system in the striatum.

C6-glioma cells endogenously express both 5HT2A receptors and inducible nitric oxide synthase (iNOS). iNOS can be induced by transcriptional activation to produce nitric oxide (NO) in response to a challenge with lipopolysaccharide (LPS). Experiments were conducted to determine whether 5HT2A receptor activation could modify the production of NO in response to LPS. Incubation of 10 microg/ml LPS with C6-glioma cells for a period of 24 hours resulted in a 2.6 fold increase in nitrite levels, as a measure of NO levels, over vehicle treated controls. Co-incubation with the selective 5HT2A receptor partial agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine (DOI) produced a dose-dependent inhibition of the LPS-induced nitrite levels of 22% with an IC50 of 16 nM. The full agonists serotonin (5HT) and alpha-methyl-5HT produced an inhibition of approximately 30% at a concentration of 1 microM. The inhibitory effect of 1 microM DOI was blocked by the 5HT2A receptor antagonists spiperone and ritanserin (10 nM). Inhibition of protein kinase C (PKC) using 100 nM chelerythrine prevented the DOI-mediated decrease in LPS-induced nitrite levels. Addition of DOI to the cells after 1 hr following the LPS addition did not produce a decrease in nitrite levels indicating iNOS was not modified post-translationally. The data demonstrate that iNOS activity can be modulated by serotonin 5HT2A receptor activation, most likely at the initiation of the induction process, via PKC. We therefore suggest that there may be a link between the serotonergic system and NO-mediated immune responses in the brain.

Serotonin potentiates pain induced by intraplantar injection of other inflammatory mediators through the 5-hydroxytryptamine2A receptor, and 5-hydroxytryptamine2A antagonists dose-dependently block pain induced by formalin. The present study examined the temporal characteristics of 5-hydroxytryptamine-mediated potentiation of pain in the rat. Simultaneous injection of the 5-hydroxytryptamine2 receptor agonist alpha-methyl-5-hydroxytryptamine (10 micrograms) plus prostaglandin E2 (0.1 microgram) produced 10 min of lifting and licking of the injected paw. When alpha-methyl-5-hydroxytryptamine was injected before prostaglandin E2, the response decreased as the interval increased, and by 1 h, it was not different from injection of prostaglandin E2 alone. When prostaglandin E2 was injected prior to alpha-methyl-5-hydroxytryptamine, sensitization to the latter persisted for more than 3 h. It was blocked by the 5-hydroxytryptamine2 antagonist ketanserin (5 micrograms), injected locally 5 min before alpha-methyl-5-hydroxytryptamine. Ketanserin (5 micrograms) or the 5-hydroxytryptamine1A/2A antagonist spiperone (0.3 microgram), injected locally 5 min before alpha-methyl-5-hydroxytryptamine and prostaglandin E2, reduced pain by 80-90%, but only by 40-50% when injected 2 min after. Formalin produced a biphasic pain response. The 5-hydroxytryptamine2 antagonists, ketanserin (50 micrograms), spiperone (3 micrograms) and ritanserin (50 micrograms) injected 5 min before formalin reduced the pain response by 70, 90 and 74%, respectively, but only by 40, 30 and 24% when injected at the beginning of the second phase. Pretreatment of the paw with 2 micrograms indomethacin did not alter the response to alpha-methyl-5-hydroxytryptamine plus prostagladin E2, indicating that the sensitization was not due to prostaglandin synthesis. These data show that 5-hydroxytryptamine sensitizes tissue to the pain-producing actions of other inflammatory mediators, and that the sensitization produces a persisting change in tissue that can be blocked by pretreatment with an antagonist, but not reversed after it has occurred. The data imply that 5-hydroxytryptamine2A antagonists may act prophylactically to prevent the evolution of pain in injured tissue, but do not reduce already-present pain.

The herbicide atrazine (ATR) has a potential toxic effect on the neuronal circuits of the brain, specifically on two major dopaminergic pathways: the nigrostriatal and mesolimbic circuits. In this work, we repeatedly exposed adult male Sprague-Dawley rats to 6 injections of 100 mg ATR/kg of body weight (for two weeks) and one saline injection two days after ATR administration. Locomotor activity was assessed for 15 minutes and/or 2 hours after ATR or saline injection and 2 months after the final ATR administration. The specific binding of [3H]-SCH23390 to D1-DA receptors and that of [3H]-Spiperone to D2-DA receptors in the dorsal and ventral striatum were assessed 2 days and 2 months after ATR treatment. ATR administration resulted in immediate, short- and long-term hypoactivity and reduced specific binding of [3H]-SCH23390 in the dorsal striatum of rats evaluated 2 months after the last ATR injection. The specific binding of [3H]-SCH23390 in the ventral striatum and the specific binding of [3H]-Spiperone in the dorsal and ventral striatum remained unchanged at 2 days or 2 months after ATR treatment. These results, together with previous findings of our group, indicate that the nigrostriatal system is a preferential target for ATR exposure.