High content phenotypic screening identifies serotonin receptor modulators with selective activity upon breast cancer cell cycle and cytokine signaling pathways.

Heterogeneity in disease mechanisms between genetically distinct patients contributes to high attrition rates in late stage clinical drug development. New personalized medicine strategies aim to identify predictive biomarkers which stratify patients most likely to respond to a particular therapy. However, for complex multifactorial diseases not characterized by a single genetic driver, empirical approaches to identifying predictive biomarkers and the most promising therapies for personalized medicine are required. In vitro pharmacogenomics seeks to correlate in vitro drug sensitivity testing across panels of genetically distinct cell models with genomic, gene expression or proteomic data to identify predictive biomarkers of drug response. However, the vast majority of in vitro pharmacogenomic studies performed to date are limited to dose-response screening upon a single viability assay endpoint. In this article we describe the application of multiparametric high content phenotypic screening and the theta comparative cell scoring method to quantify and rank compound hits, screened at a single concentration, which induce a broad variety of divergent phenotypic responses between distinct breast cancer cell lines. High content screening followed by transcriptomic pathway analysis identified serotonin receptor modulators which display selective activity upon breast cancer cell cycle and cytokine signaling pathways correlating with inhibition of cell growth and survival. These methods describe a new evidence-led approach to rapidly identify compounds which display distinct response between different cell types. The results presented also warrant further investigation of the selective activity of serotonin receptor modulators upon breast cancer cell growth and survival as a potential drug repurposing opportunity.

Structural requirements for the antitubercular quaternized triflupromazine pharmacophore.

Quaternized triflupromazine derivatives (QTDs) must possess benzyl groups attached to the quaternary nitrogen in order to have significant antitubercular potency. Replacing the quaternary amine with a triazole abolishes antitubercular activity. A modest halogen substitution effect exists, with the 4-bromophenyl QTD 3 having the best selectivity index (>21). All N-benzyl QTDs 1-4 similarly inhibit non-replicating, persistent Mycobacterium tuberculosis with MIC<8 µM, and compounds 1-3 were all nontoxic to mammalian cells in vitro (IC(50)>128 µM).

The identification of inhibitors of Schistosoma mansoni miracidial transformation by incorporating a medium-throughput small-molecule screen.

In Schistosoma mansoni, the miracidium-to-primary sporocyst transformation process is associated with many physiological, morphological, transcriptional and biochemical changes. In the present study, we use a medium-throughput small-molecule screen to identify chemical compounds inhibiting or delaying the in vitro transformation of miracidia to the sporocyst stage. The Sigma-Aldrich Library of Pharmacologically Active Compounds (LOPAC) contains 1280 well-characterized chemical compounds with various modes of action including enzyme inhibitors, antibiotics, cell-cycle regulators, apoptosis inducers and GPCR ligands. We identified 47 compounds that greatly reduce or delay this transformation process during a primary screen of live miracidia. The majority of compounds inhibiting larval transformation were from dopaminergic, serotonergic, ion channel and phosphorylation classes. Specifically, we found that dopamine D2-type antagonists, serotonin reuptake inhibitors, voltage-gated calcium channel antagonists and a PKC activator significantly reduced in vitro miracidial transformation rates. Many of the targets of these compounds regulate adenylyl cyclase activity, with the inhibition or activation of these targets resulting in increased cAMP levels in miracidia and concomitant blocking/delaying of larval transformation.

Effects of chemical manipulation of mitotic arrest and slippage on cancer cell survival and proliferation.

Microtubule-targeting cancer therapies interfere with mitotic spindle dynamics and block cells in mitosis by activating the mitotic checkpoint. Cells arrested in mitosis may remain arrested for extended periods of time or undergo mitotic slippage and enter interphase without having separated their chromosomes. How extended mitotic arrest and mitotic slippage contribute to subsequent cell death or survival is incompletely understood. To address this question, automated fluorescence microscopy assays were designed and used to screen chemical libraries for modulators of mitotic slippage. Chlorpromazine and triflupromazine were identified as drugs that inhibit mitotic slippage and SU6656 and geraldol as chemicals that stimulate mitotic slippage. Using the drugs to extend mitotic arrest imposed by low concentrations of paclitaxel led to increased cell survival and proliferation after drug removal. Cells arrested at mitosis with paclitaxel or vinblastine and chemically induced to undergo mitotic slippage underwent several rounds of DNA replication without cell division and exhibited signs of senescence but eventually all died. By contrast, cells arrested at mitosis with the KSP/Eg5 inhibitor S-trityl-L-cysteine and induced to undergo mitotic slippage were able to successfully divide and continued to proliferate after drug removal. These results show that reinforcing mitotic arrest with drugs that inhibit mitotic slippage can lead to increased cell survival and proliferation, while inducing mitotic slippage in cells treated with microtubule-targeting drugs seems to lead to protracted cell death.

Inhibitors of clathrin-dependent endocytosis enhance TGFbeta signaling and responses.

Clathrin-dependent endocytosis is believed to be involved in TGFbeta-stimulated cellular responses, but the subcellular locus at which TGFbeta induces signaling remains unclear. Here, we demonstrate that inhibitors of clathrin-dependent endocytosis, which are known to arrest the progression of endocytosis at coated-pit stages, inhibit internalization of cell-surface-bound TGFbeta and promote colocalization and accumulation of TbetaR-I and SARA at the plasma membrane. These inhibitors enhance TGFbeta-induced signaling and cellular responses (Smad2 phosphorylation/nuclear localization and expression of PAI-1). Dynasore, a newly identified inhibitor of dynamin GTPase activity, is one of the most potent inhibitors among those tested and, furthermore, is a potent enhancer of TGFbeta. Dynasore ameliorates atherosclerosis in the aortic endothelium of hypercholesterolemic ApoE-null mice by counteracting the suppressed TGFbeta responsiveness caused by the hypercholesterolemia, presumably acting through its effect on TGFbeta endocytosis and signaling in vascular cells.

The effect of calmodulin antagonists on experimental scoliosis: a pinealectomized chicken model.

STUDY DESIGN: Randomized controlled. OBJECTIVE: To evaluate the effects of Tamoxifen (TMX) and trifluoperozine (TFP) on pinealectomized chicken scoliosis. SUMMARY OF BACKGROUND DATA: Pinealectomized chicken develops scoliosis probably due to the lack of melatonin. In addition to other functions, melatonin also acts as a calmodulin antagonist. We postulate that loss of this antagonistic effect may be the cause of scoliosis in this model. TMX and TFP are known calmodulin antagonists, which may alter the incidence and severity of scoliosis. METHODS: Seventy-two newly hatched chicken that underwent surgical pinealectomy within 72 hours of hatching were divided into 3 groups of 24 animals in each as group I (control), group II (TMX), and group III (TFP). TMX and TFP were given to groups II and III, respectively, for 10 weeks with the dose of 0.1 mg/kg/d, whereas the control group received no medication. AP scoliosis radiographs were obtained at seventh and 10th week to evaluate coronal spinal alignment. RESULTS: Three chickens in group I, 2 chickens in group II, and 1 chicken in group III died in the first postoperative week. Scoliosis incidences and magnitudes were similar among groups at seventh and 10th week. TMX and TFP groups showed decreases of incidence of upper cervical, lower cervical, lower cervical-thoracic-lumbar curves at 10th week compared with seventh week. TMX group showed a decline in thoracic region mean Cobb angle, whereas control group showed an increase (P = 0.048). TMX group showed a more prominent decline in cervicothoracic region mean Cobb angle compared with control group (P = 0.009). CONCLUSION: The incidence and magnitude of scoliosis in pinealectomized chicken may be decreased by the administration of TMX, presumably because of this drugs' calmodulin antagonism. Further studies on higher animals and dosage and timing are required.

Synthesis and antitubercular activity of quaternized promazine and promethazine derivatives.

Quaternized chlorpromazine, triflupromazine, and promethazine derivatives were synthesized and examined as antitubercular agents against both actively growing and non-replicating Mycobacterium tuberculosis H37Rv. Impressively, several compounds inhibited non-replicating M. tuberculosis at concentrations equal to or double their MICs against the actively growing strain. All active compounds were non-toxic toward Vero cells (IC50 > 128 microM). N-Allylchlorpromazinium bromide was only weakly antitubercular, but replacing allyl with benzyl or substituted benzyl improved potency. An electron-withdrawing substituent on the phenothiazine ring was also essential. Branching at the carbon chain decreased antitubercular activity. The optimum antitubercular structures possessed N-(4- or 3-chlorobenzyl) substitution on triflupromazine.

Triflupromazine: a microbicide non-antibiotic compound.

The antipsychotic phenothiazine triflupromazine, possessing a methyl-thio substituent at position 10 and a fluorine moiety at position 2, exhibited significant antibacterial activity against 279 strains of Gram-positive and Gram-negative bacteria. The minimum inhibitory concentration (MIC) of the drug, according to the agar dilution method, was between 2 and 50 microg/ml for Staphylococcus aureus, and 5 and 100 microg/ml for shigellae and vibrios. Triflupromazine, when injected intraperitoneally into Swiss albino mice at a concentration of 30 microg/mouse (20 g), manifested a significant protection to the mice (p<0.001) when they were challenged with 50 median lethal dose (MLD) of Salmonella typhimurium NCTC 74. Moreover, there was a statistically significant reduction in the number of viable bacteria in organ homogenates and blood of mice treated with this phenothiazine compound.

Inhibitory and lytic effects of phenothiazine derivatives and related tricyclic neuroleptic compounds, on Entamoeba histolytica HK9 and HM1 trophozoites.

It has been shown previously that tricyclic neuroleptics like clomipramine and chlorpromazine have lethal effects on Leishmania donovani and L. major, and other studies indicate that the phenothiazine inhibitors of trypanothione reductase are potential anti-trypanosomal and anti-leishmanial drugs. With this in mind and our original observation on the presence of trypanothione in Entamoeba histolytica HK9, we examined the possible inhibitory effects of various phenothiazine and tricyclic derivatives on this human parasite. We found that drugs like clomipramine (KD002), the most potent in vitro inhibitor of trypanothione reductase among 30 tricyclic compounds tested, at 25 microM after 24 h of culture under aerobic conditions, caused a substantial decrease in the number of E. histolytica HK9 trophozoites, from approx. 15 x 10(6) to 5.37 x 10(6) cells, and at 100 microM to 0.8 x 10(6) cells. A substantial inhibitory effect on cell proliferation could also be demonstrated with metronidazol (used clinically against amoebiasis). Under similar experimental conditions other tricyclic and phenothiazine derivatives (OFKs), designed originally to inhibit the trypanothione reductase of trypanosomatides, had an inhibitory effect of 16 to 95%. For comparison, similar results were obtained using clomipramine and a phenothiazine derivative (OFK006) with Trypanosoma cruzi and Crithidia luciliae, except that with the latter the inhibitory effect of clomipramine was less dramatic. Experiments comparing two E. histolytica strains showed that normal cell proliferation under anaerobiosis was higher in strain HK9 than in HM1, which is highly virulent, but that metronidazol and clomipramine were less effective against HM1. Two other drugs tested, diphenydramine (KD005) and a phenothiazine derivative (OFK008), also had significant but lower inhibitory effects on both strains. The inhibitory activity on cell proliferation and the lytic effects on this human parasite by the tricyclic compounds clomipramine, chlorpromazine and others, as well as by the phenothiazine derivatives, indicate that they can be considered potential anti-amoebic agents.

Structure-activity relationships of phenothiazines in inhibiting lymphocyte motility as determined by a novel flow cytometric assay.

Lymphocyte motility is highly dependent on rapid changes in cell shape. The human T-lymphoma cell line, MOLT-4, is constitutively shape-changing and motile, and both of these properties can be inhibited by the phenothiazine, chlorpromazine, as assessed by video analysis and migration across polycarbonate filters. In this paper, the light-scattering facility of a flow cytometer has been used to establish a simpler and more quantitative means of measuring changes in shape. By this method, the structure activity relationship (SAR) of phenothiazines and related compounds has been determined. The most active compounds had the tricyclic phenothiazine nucleus with a constrained dialkylaminoalkyl substituent at the nitrogen. The SAR for inhibition of lymphocyte motility differs from those reported for neuroleptic effects and for inhibition of PKC or calmodulin. Phenothiazine concentrations that inhibited lymphocyte shape-changing resulted in reduced F-actin concentrations. This indicates that the probable mode of action is disruption of mechanisms regulating actin polymerisation.

Trifluopromazine late preventive effects on carbon tetrachloride-induced liver necrosis.

Trifluopromazine (TFPro) administration to rats (50 mg/kg, ip) 30 min before or 6 or 10 hr after CCl4 treatment (1 ml/kg ip in olive oil) partially prevented necrogenic effects of this compound at 24 hr. TFPro has only minor effects on the covalent binding (CB) of CCl4-reactive metabolites to cellular constituents and even an enhancing action on CCl4-promoted lipid peroxidation (LP). Determination of TFPro levels in liver 1 and 3 hr after administration by gas chromatography/mass spectrometry showed its presence in that tissue at concentrations well above those needed for calmodulin (CaM) inhibitory effects of this drug. TFPro lowered body temperature in CCl4-treated animals during the 24-hr observation period. Protective effects of TFPro at 6 or 10 hr, when most of the CB and all of the LP has already occurred, suggest but do not prove a role for CaM in late stages of CCl4-induced necrogenic effects. Decreases in the body temperature of CCl4-poisoned animals provoked by TFPro might also play a role in the preventive actions of this drug.

[Phenothiazine derivatives, chlorpromazine and triflupromazine, produce different effects on sympathetic nerve activity in urethane-anesthetized rabbits].

UNLABELLED: This study was designed to evaluate effects of chlorpromazine (CPZ) or triflupromazine (TPZ) on renal sympathetic nerve activity and hemodynamics in urethane-anesthetized rabbits. METHODS: Thirty-five rabbits were divided into the following two groups: CPZ group (N = 10) and TPZ group (N = 25), challenged by an intravenous injection of either CPZ (0.4 mg.kg-1) or TPZ (0.2 mg.kg-1), respectively. Each experimental group was further divided into the following groups. CPZ group was divided into CPZ-INTACT group of animals with neuraxis intact (N = 6) and CPZ-SAD group of animals with combined denervation of the carotid sinus and aortic nerves (N = 4). TPZ group was divided into TPZ-INTACT group of rabbits with neuraxis intact (N = 8), TPZ-VAGOTOMY group of only cervical vagotomy (N = 6), TPZ-SADV group with combined denervation of the carotid sinus and aortic nerves with cervical vagotomy (N = 5) and TPZ-VI group with right cervical vagotomy but having the intact left vagus (N = 6). In the last group, right afferent vagal nerve activity was measured simultaneously. Mean blood pressure, central venous pressure, heart rate and renal sympathetic nerve activity were measured at the same time. RESULTS: In the CPZ-INTACT group, the agent caused a decrease in mean blood pressure and significant increases in sympathetic nerve activity and heart rate, but no significant change in central venous pressure. This increase in sympathetic nerve activity and heart rate disappeared in the SAD animals in spite of hypotension. Animals of the TPZ-INTACT group showed an abrupt decrease in sympathetic nerve activity in response to hypotension, but did not exhibit a remarkable change in heart rate and central venous pressure. However, in contrast, animals with severed cervical vagi showed a sympathetic augmentation after the TPZ injection. These sympathetic changes were abolished in the SADV animals. In the TPZ-VI group, TPZ elicited a decline in sympathetic nerve activity similar to that observed in INTACT animals, but afferent vagal nerve activity increaed simultaneously with sympathetic depression. CONCLUSION: These results indicate that a reflex increase in sympathetic nerve activity which occurred during hypotension after CPZ injection may have been mediated by the arterial baroreceptor reflex and that a sympathetic reduction after TPZ administration may have resulted from a reciprocal interaction between an excitation through the sino-aortic nerves and an inhibition via the vagal nerves.

Comparative studies of phenothiazine derivatives for their effects on swelling of normal and sickle erythrocytes.

1. Three phenothiazines with similar structure; chlorpromazine (CPZ), triflupromazine (TFPZ), and trifluoperazine (TFP), were compared for the potency of swelling of normal red cells and antisickling effect of red cell from patients with sickle cell disease. 2. Normal erythrocytes treated with the phenothiazines became swollen within 60 min, and the percent increase in mean cell volume (MCV) was dose-dependent and varied according to the agent used. 3. The cell swelling was hematocrit-dependent, and pH-dependent. A greater swelling potency was seen at lower hematocrits and higher pH values. 4. The swelling was also dependent on the pKa values of these chemicals. TFP with the lowest pKa induced the highest degree of swelling while CPZ with the highest pKa induced the lowest. 5. The un-ionized fraction of the phenothiazines at a given pH was directly related to potency of the swelling. 6. The cell swelling was directly related to the binding affinities of the phenothiazines to calmodulin. 7. The antisickling effect of these compounds using sickle red cells, numerically estimated by an automated image analysis system, was found in the same order as that of swelling potency: TFP > TFPR > CPZ.

Killing of endothelial cells and release of arachidonic acid. Synergistic effects among hydrogen peroxide, membrane-damaging agents, cationic substances, and proteinases and their modulation by inhibitors.

51Chromium-labeled rat pulmonary artery endothelial cells (EC) cultivated in MEM medium were killed, in a synergistic manner, by mixtures of subtoxic amounts of glucose oxidase-generated H2O2 and subtoxic amounts of the following agents: the cationic substances, nuclear histone, defensins, lysozyme, poly-L-arginine, spermine, pancreatic ribonuclease, polymyxin B, chlorhexidine, cetyltrimethyl ammonium bromide, as well as by the membrane-damaging agents phospholipases A2 (PLA2) and C (PLC), lysolecithin (LL), and by streptolysin S (SLS) of group A streptococci. Cytotoxicity induced by such mixtures was further enhanced by subtoxic amounts either of trypsin or of elastase. Glucose-oxidase cationized by complexing to poly-L-histidine proved an excellent deliverer of membrane-directed H2O2 capable of enhancing EC killing by other agonists. EC treated with rabbit anti-streptococcal IgG were also killed, in a synergistic manner, by H2O2, suggesting the presence in the IgG preparation of cross-reactive antibodies. Killing of EC by the various mixtures of agonists was strongly inhibited by scavengers of hydrogen peroxide (catalase, dimethylthiourea, MnCl2), by soybean trypsin inhibitor, by polyanions, as well as by putative inhibitors of phospholipases. Strong inhibition of cell killing was also observed with tannic acid and by extracts of tea, but less so by serum. On the other hand, neither deferoxamine, HClO, TNF, nor GTP gamma S had any modulating effects on the synergistic cell killing. EC exposed either to 6-deoxyglucose, puromycin, or triflupromazin became highly susceptible to killing by mixtures of hydrogen peroxide with several of the membrane-damaging agents. While maximal synergistic EC killing was achieved by mixtures of H2O2 with either PLA2, PLC, LL, or with SLS, a very substantial release of [3H]arachidonic acid (AA), PGE2, and 6-keto-PGF occurred only if a proteinase was also added to the mixture of agonists. The release of AA from EC was markedly inhibited either by scavengers of H2O2, by proteinase inhibitors, by cationic agents, by HClO, by tannic acid, and by quinacrin. We suggest that cellular injury induced in inflammatory and infectious sites might be the result of synergistic effects among leukocyte-derived oxidants, lysosomal hydrolases, cytotoxic cationic polypeptides, proteinases, and microbial toxins, which might be present in exudates. These "cocktails" not only kill cells, but also solubilize AA and several of its metabolites. However, AA release by the various agonists can be also achieved following attack by leukocyte-derived agonists on dead cells. It is proposed that treatment by "cocktails" of adequate antagonists might be beneficial to protect against cellular injury in vivo.

Trypanosoma cruzi: effect of phenothiazines on the parasite and its interaction with host cells.

Phenothiazines were observed to have a direct effect on Trypanosoma cruzi and on its in vitro interaction with host cells. They caused lysis of trypomastigotes (50 uM/24 h) and, in axenic medium, dose-dependent inhibition of amastigote and, to a lesser extent, epimastigote proliferation. Treatment of infected peritoneal macrophages with 12.5 uM chlorpromazine or triflupromazine inhibited the infection; this effect was found to be partially reversible if the drugs were removed after 24 h of treatment. At 60 uM, the drugs caused damage to amastigotes interiorized in heart muscle cells. However, the narrow margin of toxicity between antitrypanosomal activity and damage to host cells mitigates against in vivo investigation at the present time. Possible hypotheses for the mechanism of action of phenothiazines are discussed.

Evaluation of triflupromazine as a sedative in camels (Camelus dromedarius).

Effects of administration of triflupromazine were evaluated in 11 adult domesticated camels (Camelus dromedarius) weighing 403 +/- 29.5 kg (Mean +/- SE). Six camels were used to evaluate sedative properties of the drug and its effects on haematological and blood biochemical parameters. In the remaining 5 camels, effects on haemodynamics, acid base status and blood gases were studied. In all the animals triflupromazine was administered intramuscularly in the gluteal region at the rate of 2 mg/kg. Camels voluntarily sat down 48.9 +/- 5.4 min after administration of the drug but stood up again if disturbed. Drowsiness, drooping of lower lip and salivation were evident. The animals stood on their own and started walking with ataxia after 159 +/- 7 min and recovered completely from the effect of drug within 259 +/- 23 min. The drug caused a significant tachycardia and a moderate hypotension. The decrease in central venous pressure was also significant. Rectal temperature, respiratory rate, acid base status, blood gases, haemoglobin concentration, packed cell volume, total erythrocyte count, total leucocyte count, differential leukocyte count, blood urea nitrogen, plasma alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, alkaline phosphatase, blood glucose and plasma concentrations of sodium, potassium, chloride and inorganic phosphate were not significantly affected by triflupromazine.

Modification of radiation induced lipid peroxidation by calmodulin antagonists.

It has been shown that calmodulin antagonists provide radio-protection in euoxic and sensitization in hypoxic conditions. This differential protection in euoxic conditions might have arisen from the interaction of calmodulin antagonists with oxygen free radicals. This possibility has been tested in the present communication. Radiation induced lipid peroxidation process in liposomes has been used for this purpose. Liposomes prepared from L-alpha-lecithin were irradiated with or without calmodulin antagonists. Calmodulin antagonists inhibited lipid peroxidation significantly. The inhibition was found to increase with increase in concentration of the drugs. These observations suggest that calmodulin antagonists have a capacity to scavenge oxygen free radicals involved in initiation and/or propagation of lipid peroxidation process. This may be the reason for their differential radioprotection in euoxic conditions in biological systems.

Effects of calmodulin antagonists on human adenoidal mast cells.

N-(6-aminohexyl)-1-naphthalenesulfonamide (W5), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) and Triflupromazine (TFPr) are substances with calmodulin antagonistic properties. All of these compounds inhibited the Con A-induced histamine release from human adenoidal mast cells. Maximal inhibition for W5 was observed at 200 microM, for W7 at 50 microM and for TFPr at 20 microM. Higher concentrations of each substance induced a marked histamine release. The kinetics of the mediator release were found to be different for Con A and W7 only after 5 mins incubation. The kinetics of TFPr were found to be biphasic: a slow onset was followed by a fast release reaction.

[The effects of PGI2 analog (OP-41483) on hemodynamics and myocardial metabolism during simple deep hypothermia].

The effects of PGI2 analog (OP-41483) on hemodynamics and myocardial metabolism were compared with those of triflupromazine in dogs under simple deep hypothermia. Twenty dogs were divided into two groups according to the agent administered. In group OP (n = 10), intravenous administration of OP-41483 was performed continuously during the procedure. While the bolus of intravenous triflupromazine was injected at the beginning of cooling in group TR (n = 10). 1) Systemic and coronary vascular resistance decreased during cooling period in group OP but it increased in group TR. 2) Myocardial lactate extraction ratio was higher in group OP compared with group TR, but the ratio remained above 10% in both groups. 3) No significant differences in mean coronary blood flow and myocardial oxygen consumption were noted between the two groups. The study suggests that OP-41483 is one of the useful agents to maintain coronary circulation and myocardial metabolism under simple deep hypothermia.