Chlorpromazine oligomer is a potentially active substance that inhibits human D-amino acid oxidase,product of a susceptibility gene for schizophrenia

D-Amino acid oxidase (DAO), a potential risk factor for schizophrenia, has been proposed to be involved in the decreased glutamatergic neurotransmission in schizophrenia. Here we show the inhibitory effect of an antipsychotic drug, chlorpromazine, on human DAO, which is consistent with previous reports using porcine DAO, although human DAO was inhibited to a lesser degree (K_i = 0.7 mM) than porcine DAO. Since chlorpromazine is known to induce phototoxic or photoallergic reactions and also to be transformed into various metabolites, we examined the effects of white light-irradiated chlorpromazine on the enzymatic activity. Analytical methods including high-resolution mass spectrometry revealed that irradiation triggered the oligomerization of chlorpromazine molecules. The oligomerized chlorpromazine showed a mixed type inhibition with inhibition constants of low micromolar range, indicative of enhanced inhibition. Taken together, these results suggest that oligomerized chlorpromazine could act as an active substance that might contribute to the therapeutic effects of this drug.     

Chlorpromazine oligomer is a potentially active substance that inhibits human D-amino acid oxidase, product of a susceptibility gene for schizophrenia

D-amino acid oxidase (DAO), a potential risk factor for schizophrenia, has been proposed to be involved in the decreased glutamatergic neurotransmission in schizophrenia. Here we show the inhibitory effect of an antipsychotic drug, chlorpromazine, on human DAO, which is consistent with previous reports using porcine DAO, although human DAO was inhibited to a lesser degree (K(i) = 0.7 mM) than porcine DAO. Since chlorpromazine is known to induce phototoxic or photoallergic reactions and also to be transformed into various metabolites, we examined the effects of white light-irradiated chlorpromazine on the enzymatic activity. Analytical methods including high-resolution mass spectrometry revealed that irradiation triggered the oligomerization of chlorpromazine molecules. The oligomerized chlorpromazine showed a mixed type inhibition with inhibition constants of low micromolar range, indicative of enhanced inhibition. Taken together, these results suggest that oligomerized chlorpromazine could act as an active substance that might contribute to the therapeutic effects of this drug.     

Antischizophrenic drugs: differential plasma protein binding and therapeutic activity.

The percentage of free neuroleptic drug unbound to plasma protein is much higher for the respective metabolites of chlorpromazine and thioridazine, 7-hydroxychlorpromazine and mesoridazine, than for the parent drugs. The therapeutic activities of chlorpromazine and thioridazine may be mediated to a major extent by 7-hydroxychlorpromazine and mesoridazine respectively. Measuring free levels of the active metabolites of neuroleptics as well as the parent drugs may facilitate regulation of neuroleptic doses to secure optimal therapeutic responses.     

Discovery of Cellular Proteins Required for the Early Steps of HCV Infection Using Integrative Genomics

Successful viral infection requires intimate communication between virus and host cell, a process that absolutely requires various host proteins. However, current efforts to discover novel host proteins as therapeutic targets for viral infection are difficult. Here, we developed an integrative-genomics approach to predict human genes involved in the early steps of hepatitis C virus (HCV) infection. By integrating HCV and human protein associations, co-expression data, and tight junction-tetraspanin web specific networks, we identified host proteins required for the early steps in HCV infection. Moreover, we validated the roles of newly identified proteins in HCV infection by knocking down their expression using small interfering RNAs. Specifically, a novel host factor CD63 was shown to directly interact with HCV E2 protein. We further demonstrated that an antibody against CD63 blocked HCV infection, indicating that CD63 may serve as a new therapeutic target for HCV-related diseases. The candidate gene list provides a source for identification of new therapeutic targets.     

A comparison of the effects of chlorpromazine, 7-hydroxychlorpromazine and chlorpromazine sulfoxide on the activity of central dopaminergic neurons

Using single unit recording techniques, chlorpromazine and two of its naturally occuring metabolites in man, 7-hydroxychlorpromazine and chlorpromazine sulfoxide, were tested for their ability to reverse amphetamine-induced depression of rat dopaminergic ventral tegmental neurons (A10). Small equivalent doses of chlorpromazine and 7-hydroxychlorpromazine were found to readily reverse amphetamine-induced depression of these cells. Chlorpromazine sulfoxide was found to be 50–100 times less potent in this regard. Previous findings have demonstrated that only phenothiazines with antipsychotic properties reverse amphetamine-induced depression of A10 neurons. Thus, we would predict that 7-hydroxychlorpromazine would have anti-psychotic properties whereas chlorpromazine sulfoxide would not. A preliminary study by Sakalis et al., suggests that plasma levels of chlorpromazine and 7-hydroxychlorpromazine are possibly correlated with the therapeutic effects of chlorpromazine in schizophrenia. Chlorpromazine sulfoxide levels, on the other hand, are reported to be high in chlorpromazine treated non-responders. Thus there is a direct parallel between predictions of antipsychotic efficacy based on our test model and the possible clinical importance of these chlorpromazine metabolites. Both findings suggest that 7-hydroxychlopromazine might be a good antipsychotic agent.     

Innate immunity against Leishmania infections

Leishmaniasis is a major health problem that affects more than 300 million people throughout the world. The morbidity associated with the disease causes serious economic burden in Leishmania endemic regions. Despite the morbidity and economic burden associated with Leishmaniasis, this disease rarely gets noticed and is still categorized under neglected tropical diseases. The lack of research combined with the ability of Leishmania to evade immune recognition has rendered our efforts to design therapeutic treatments or vaccines challenging. Herein, we review the literature on Leishmania from innate immune perspective and discuss potential problems as well as solutions and future directions that could aid in identifying novel therapeutic targets to eliminate this parasite.     

Chlorpromazine-induced changes of endocytosis in blastomeres of the embryos of pond snail <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis> L. and Eurasian weather loach <Emphasis Type="Italic">Misgurnus fossilis</Emphasis> L.

Chlorpromazine causes distinct defects in normal development of early cleaving embryos of gastropod pond snail (Lymnaea stagnalis L.) and teleost Eurasian weather loach (Misgurnus fossilis L.): a decrease in blastomere contact surfaces following the rounding in gastropod embryo and a reduction of cleaving blastodisk surface in teleost. Right after the application of chlorpromazine fluorescence is visible as bright spots on the surface of blastomeres. Then spherical vesicles appear within the whole volume of the cells. In loach, association of chlorpromazine with the plasma membrane was observed in blastodisk but not in the yolk cell membrane. Electron microscopy has shown that chlorpromazine induces folding of the cell membrane. Experiments with fluorescent dextran probe demonstrate that chlorpromazine modifies the rate and character of the dextran uptake. Our results indicate that chlorpromazine binds to specific sites of plasmalemma and stimulates endocytosis. Patterns of chlorpromazine binding to the membrane and its effects on endocytosis are similar in teleost and gastropod.     

Preventive and therapeutic vaccination with PAP-3, a novel human prostate cancer peptide, inhibits carcinoma development in HLA transgenic mice

Conventional treatment of recurrent and metastasized prostate cancer (CaP) remains inadequate; this fact mandates development of alternative therapeutic modalities, such as specific active or passive immunotherapy. Previously, we reported the identification of a novel highly immunogenic HLA-A*0201-restricted Prostatic Acid Phosphatase-derived peptide (PAP-3) by a two-step in vivo screening in an HLA-transgenic (HHD) mouse system. In the present study we aimed at elucidating the efficiency of PAP-3-based vaccine upon active antitumor immunization. To this end we established preventive and therapeutic carcinoma models in HHD mice. The 3LL murine Lewis lung carcinoma clone D122 transduced to express HLA-A*0201 and PAP served as a platform for these models. The HLA-A*0201–PAP-3 complex specific recombinant single chain scFV-PAP-3 antibodies were generated and used to confirm an endogenous PAP processing resulting in PAP-3 presentation by HLA-A*0201. PAP-3 based vaccines significantly decreased tumor incidence in a preventive immunization setting. Therapeutic vaccination of HHD mice with PAP-3 led to rejection of early established tumors and to increase of mouse survival. These results strongly support a therapeutic relevance of the identified CTL epitope upon active antitumor immunization. The newly established carcinoma model presented herein might be a useful tool for cancer vaccine design and optimization.     

A genetic view of laryngeal cancer heterogeneity

During the recent decades significant improvements in the understanding of laryngeal molecular biology allowed a better characterization of the tumor. However, despite increased molecular knowledge and clinical efforts, survival of patients with laryngeal cancer remains the same as 30 years ago. Although this result may not make major conclusions as preservation approaches were not broadly used until the time of database collection, it seems to be clear that there is still window for improvement. Although the cornerstone for laryngeal cancer eradication is to implement smoking cessation programs, survival progresses will be hopefully seen in the future. Introducing molecular biomarkers as predictive factors to determine which patients will benefit of preservation treatments may become one of the next steps to improve survival. Furthermore, the development of new therapeutic modalities joint to biomarkers to selectively apply such new therapy in these patients may help to define new modalities with improved survival. New inhibitors against Notch pathway, EGFR, VRK1 or DNA damage repair may become gold standard if we are able to identify patients that may benefit from them, either on survival or functional larynx preservation. It is the moment for an inflexion point on the way laryngeal cancer is clinically managed.     

A Calcium/Calmodulin-regulated Member of the Receptor-like Kinase Family Confers Cold Tolerance in Plants

Cold is a limiting environmental factor that adversely affects plant growth and productivity. Calcium/calmodulin-mediated signaling is believed to play a pivotal role in plant response to cold stress, but its exact role is not clearly understood. Here, we report that CRLK1, a novel calcium/calmodulin-regulated receptor-like kinase, is crucial for cold tolerance in plants. CRLK1 has two calmodulin-binding sites with different affinities as follows: one located at residues 369–390 with a K_d of 25 nm, and the other located at residues 28–112 with a K_d of 160 nm. Calcium/calmodulin stimulated the kinase activity, but the addition of chlorpromazine, a calmodulin antagonist, blocked its stimulation. CRLK1 is mainly localized in the plasma membrane, and its expression is stimulated by cold and hydrogen peroxide treatments. Under normal growth conditions, there is no noticeable phenotypic difference between wild-type and crlk1 knock-out mutant plants. However, as compared with wild-type plants, the crlk1 knock-out mutants exhibited an increased sensitivity to chilling and freezing temperatures. Northern analysis showed that the induction of cold-responsive genes, including CBF1, RD29A, COR15a, and KIN1 in crlk1 mutants, is delayed as compared with wild-type plants. These results indicate that CRLK1 is a positive regulator of cold tolerance in plants. Furthermore, our results suggest that CRLK1 plays a role in bridging calcium/calmodulin signaling and cold signaling.     

Extended treatment with typical and atypical antipsychotic drugs differential effects on the densities of dopamine D2-like and GABAA receptors in rat striatum

In situ radioligand binding and quantitative autoradiography have been used to measure the density of striatal D1-like, D2-like, and GABAA receptors in rats treated with haloperidol at 0.01 or 0.1 mg/kg/ day or chlorpromazine, olanzapine or clozapine at 0.1 or 1.0 mg/kg/day for 1, 3 or 7 months. [3H]SCH23390 binding to D1-like receptors was not changed by any drug treatments. There were significant increases in [3H]nemonapride binding to D2-like receptors at different time points due to treatment with haloperidol, chlorpromazine and olanzapine. By contrast, treatment with clozapine and olanzapine caused a time-dependent decrease in [3H]muscimol binding to the GABAA receptor. These data suggest that treatment with atypical antipsychotic drugs, but not typical antipsychotic drugs, affect striatal GABAergic neurons. In addition, it would appear that clozapine might be unique in that it does not increase dopamine-D2 like receptor density at doses which would be predicted to have antipsychotic effects in humans. The extent to which such changes are involved in the therapeutic effects of drugs such as olanzapine and clozapine remains to be determined.     

Development of a human three-dimensional organotypic skin-melanoma spheroid model for in vitro drug testing

Despite remarkable efforts, metastatic melanoma (MM) still presents with significant mortality. Recently, mono-chemotherapies are increasingly replenished by more cancer-specific combination therapies involving death ligands and drugs interfering with cell signaling. Still, MM remains a fatal disease because tumors rapidly develop resistance to novel therapies thereby regaining tumorigenic capacity. Although genetically engineered mouse models for MM have been developed, at present no model is available that reliably mimics the human disease and is suitable for studying mechanisms of therapeutic obstacles including cell death resistance. To improve the increasing requests on new therapeutic alternatives, reliable human screening models are demanded that translate the findings from basic cellular research into clinical applications. By developing an organotypic full skin equivalent, harboring melanoma tumor spheroids of defined sizes we have invented a cell-based model that recapitulates both the 3D organization and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention. By extending our previous findings on melanoma cell sensitization toward TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) by co-application of sublethal doses of ultraviolet-B radiation (UVB) or cisplatin, we show significant differences in the therapeutical outcome to exist between regular two-dimensional (2D) and complex in vivo-like 3D models. Of note, while both treatment combinations killed the same cancer cell lines in 2D culture, skin equivalent-embedded melanoma spheroids are potently killed by TRAIL+cisplatin treatment but remain almost unaffected by the TRAIL+UVB combination. Consequently, we have established an organotypic human skin-melanoma model that will facilitate efforts to improve therapeutic outcomes for malignant melanoma by providing a platform for the investigation of cytotoxic treatments and tailored therapies in a more physiological setting.     

Mitochondria as a target for neuroprotection

Despite all the efforts of modern medical and biomedical sciences, the effective therapeutic treatments that would restore the brain functions lost after stroke have not been found yet. At the same time, experimental preclinical studies revealed an arsenal of effectors having potential for clinical applications. Identification of the key signaling pathways, both damaging and protective, can accelerate the development and implementation of new effective neuroprotectors. One of the key elements of these pathways is mitochondrion. In this context, we studied various therapeutic approaches to the treatment and prevention of cerebral ischemia, which are aimed at modulation of mitochondrial functions. The spectrum of tested neuroprotectors included antioxidants, uncouplers of respiration and phosphorylation, as well as ischemic, remote, and pharmacological preconditioning. Their efficacy and therapeutic windows were compared and the possibility of combining different methods in order to maximize their efficiency was considered.     

Fatty acid cellular metabolism and lactone production by the yeast Pichia guilliermondii

Methyl ricinoleate conversion into γ-decalactone by fungi is already widely used by the aromatic industry. It offers an interesting alternative to chemical synthesis by permitting acquisition of a natural label. Peroxisomal β-oxidation has been described as the probable transformation mechanism. This paper provides information about this metabolism and shows the importance of the step catalysed by carnitine octanoyltransferase. After culture of the yeast Pichia guilliermondii on a medium containing methyl ricinoleate as sole carbon source, we confirmed that mitochondrial β-oxidation could not be responsible for the biotransformation. We also observed the effect of chlorpromazine, an inhibitor of carnitine octanoyltransferase, on peroxisomal β-oxidation and therefore on lactone production, and on lipid accumulation by the yeasts. The presence of chlorpromazine caused a reduction in aromatic specific production yield. This reduction was inversely proportional to the amount of chlorpromazine present in the medium. A considerable accumulation of methyl ricinoleate derivatives was also observed. We therefore concluded that the metabolism responsible for the bioconversion was peroxisomal β-oxidation. The effects of chlorpromazine suggested that the entry of fatty acids into the peroxisomes took place in a carnitine-dependent manner. This step might be a limiting step in the metabolism. Received: 26 June 1995/Received revision: 16 November 1995/Accepted: 4 December 1995     

Stem cell therapies in cardiac diseases: Current status and future possibilities

Cardiovascular diseases represent the world’s leading cause of death. In this heterogeneous group of diseases, ischemic cardiomyopathies are the most devastating and prevalent, estimated to cause 17.9 million deaths per year. Despite all biomedical efforts, there are no effective treatments that can replace the myocytes lost during an ischemic event or progression of the disease to heart failure. In this context, cell therapy is an emerging therapeutic alternative to treat cardiovascular diseases by cell administration, aimed at cardiac regeneration and repair. In this review, we will cover more than 30 years of cell therapy in cardiology, presenting the main milestones and drawbacks in the field and signaling future challenges and perspectives. The outcomes of cardiac cell therapies are discussed in three distinct aspects: The search for remuscularization by replacement of lost cells by exogenous adult cells, the endogenous stem cell era, which pursued the isolation of a progenitor with the ability to induce heart repair, and the utilization of pluripotent stem cells as a rich and reliable source of cardiomyocytes. Acellular therapies using cell derivatives, such as microvesicles and exosomes, are presented as a promising cell-free therapeutic alternative.     

In vivo potencies of antipsychotic drugs in blocking alpha 1 noradrenergic and dopamine D2 receptors: implications for drug mechanisms of action

In addition to being dopamine antagonists, all antipsychotic drugs are potent antagonists of alpha-1 noradrenergic receptors. Nevertheless, the contribution of alpha blockade to the clinical therapeutic effects of the antipsychotic drugs has never attracted extensive study. In particular, the relative alpha-1 noradrenergic antagonist potency of antipsychotic drugs has rarely been determined in vivo during extended treatment, although such treatment would provide a better model of clinical drug effects than the determination of potencies in in vitro systems, such as assays of competition for binding sites in tissue homogenates, as is most often done. To estimate the physiological efficacy of antipsychotic drugs as dopamine and alpha adrenergic antagonists, we treated rats for four weeks with daily IP injections of the following antipsychotic drugs: Fluphenazine, 1 mg/kg; haloperidol, 1 mg/kg; chlorpromazine, 25 mg/kg; thioridazine, 25 mg/kg; and clozapine, 25 mg/kg. Effective antagonism should lead to an increase in density of the relevant receptors. After two drug-free days, rats were sacrificed and the affinity and density of dopamine D2 and alpha-1 noradrenergic receptors were determined in striatum and brain exclusive of striatum, respectively. Alpha 1 noradrenergic receptor density but not dopamine receptor density was increased after all treatments. Thus, preliminary experiments with this in vivo model suggest that all antipsychotic drugs are effective antagonists at alpha 1 noradrenergic receptors, while not all are effective antagonists at dopamine D2 receptors.     

Clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ACE2 with the cytoplasmic tail deleted

The penetration of various viruses into host cells is accomplished by hijacking the host endocytosis machinery. In the case of severe acute respiratory syndrome coronavirus (SARS-CoV) infection, viral entry is reported to require a low pH in intracytoplasmic vesicles; however, little is known about how SARS-CoV invades such compartments. Here we demonstrate that SARS-CoV mainly utilizes the clathrin-mediated endocytosis pathway for its entry to target cells by using infectious SARS-CoV, as well as a SARS-CoV pseudovirus packaged in the SARS-CoV envelope. The SARS-CoV entered caveolin-1-negative HepG2 cells, and the entry was significantly inhibited by treatment with chlorpromazine, an inhibitor for clathrin-dependent endocytosis, and by small interfering RNA-mediated gene silencing for the clathrin heavy chain. Furthermore, the SARS-CoV entered COS7 cells transfected with the mutant of ACE2 with the cytoplasmic tail deleted, SARS-CoV receptor, as well as the wild-type ACE2, and their entries were significantly inhibited by treatment with chlorpromazine. In addition, ACE2 translocated into EEA1-positive early endosomes immediately after the virus attachment to ACE2. These results suggest that when SARS-CoV binds ACE2 it is internalized and penetrates early endosomes in a clathrin-dependent manner and that the cytoplasmic tail of ACE2 is not required for the penetration of SARS-CoV.     

Potential Antileukemia Effect and Structural Analyses of SRPK Inhibition by N-(2-(Piperidin-1-yl)-5-(Trifluoromethyl)Phenyl)Isonicotinamide (SRPIN340)

Dysregulation of pre-mRNA splicing machinery activity has been related to the biogenesis of several diseases. The serine/arginine-rich protein kinase family (SRPKs) plays a critical role in regulating pre-mRNA splicing events through the extensive phosphorylation of splicing factors from the family of serine/arginine-rich proteins (SR proteins). Previous investigations have described the overexpression of SRPK1 and SRPK2 in leukemia and other cancer types, suggesting that they would be useful targets for developing novel antitumor strategies. Herein, we evaluated the effect of selective pharmacological SRPK inhibition by N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340) on the viability of lymphoid and myeloid leukemia cell lines. Along with significant cytotoxic activity, the effect of treatments in regulating the phosphorylation of the SR protein family and in altering the expression of MAP2K1, MAP2K2, VEGF and FAS genes were also assessed. Furthermore, we found that pharmacological inhibition of SRPKs can trigger early and late events of apoptosis. Finally, intrinsic tryptophan fluorescence emission, molecular docking and molecular dynamics were analyzed to gain structural information on the SRPK/SRPIN340 complex. These data suggest that SRPK pharmacological inhibition should be considered as an alternative therapeutic strategy for fighting leukemias. Moreover, the obtained SRPK-ligand interaction data provide useful structural information to guide further medicinal chemistry efforts towards the development of novel drug candidates.