CWHM-974 is a fluphenazine derivative with improved antifungal activity against Candida albicans due to reduced susceptibility to multidrug transporter-mediated resistance mechanisms.

Multidrug resistance (MDR) transporters such as ATP-Binding Cassette (ABC) and Major Facilitator Superfamily proteins are important mediators of antifungal drug resistance, particularly with respect to azole class drugs. Consequently, identifying molecules that are not susceptible to this mechanism of resistance is an important goal for new antifungal drug discovery. As part of a project to optimize the antifungal activity of clinically used phenothiazines, we synthesized a fluphenazine derivative (CWHM-974) with 8-fold higher activity against Candida spp. compared to the fluphenazine and with activity against Candida spp. with reduced fluconazole susceptibility due to increased MDR transporters. Here, we show that the improved C. albicans activity is because fluphenazine induces its own resistance by triggering expression of Candida drug resistance (CDR) transporters while CWHM-974 induces expression but does not appear to be a substrate for the transporters or is insensitive to their effects through other mechanisms. We also found that fluphenazine and CWHM-974 are antagonistic with fluconazole in C. albicans but not in C. glabrata, despite inducing CDR1 expression to high levels. Overall, CWHM-974 is one of the few examples of a molecule in which relatively small structural modifications significantly reduced susceptibility to multidrug transporter-mediated resistance.

Repurposing fluphenazine to suppress melanoma brain, lung and bone metastasis by inducing G0/G1 cell cycle arrest and apoptosis and disrupting autophagic flux.

Brain metastasis is the main cause of treatment failure and melanoma-related death. Inadequate concentrations of therapeutic drugs in the brain due to the blood-brain barrier (BBB) pose a major challenge in the treatment of brain metastasis. Antipsychotics can cross the BBB to reach the brain. Fluphenazine (FPZ) inhibits the survival of melanoma cells in vitro. However, its efficacy in suppressing the metastasis of melanoma, especially brain metastasis, remains unknown. Therefore, we explored whether fluphenazine (FPZ) can be repurposed for treating melanoma metastasis. A subcutaneous tumor model, and experimental metastasis models that simulate the outgrowth of melanoma cells in the brain, lung, and bone were established to verify the inhibitory effect of FPZ on melanoma cells. FPZ showed potential inhibitory effects against melanoma both in vivo and in vitro. It induced G0/G1 phase arrest and-mitochondrion-mediated intrinsic apoptosis, and inhibited autophagic flux in melanoma cells in vitro. In vivo, subcutaneous tumor, brain, lung, and bone models of metastatic melanoma were established. Intraperitoneal injection of FPZ (8 mg/kg) significantly inhibited melanoma growth in the subcutaneous and experimental metastasis models. In a lung metastasis model, FPZ reduced the proportion of M2 macrophages and increased the proportion of CD8+ T cells and NK cells in vivo, thereby promoting an anticancer immune response. The findings of this study indicate that FPZ is a potential drug candidate for treating metastatic melanoma.

Combination of Antimalarial and CNS Drugs with Antineoplastic Agents in MCF-7 Breast and HT-29 Colon Cancer Cells: Biosafety Evaluation and Mechanism of Action.

Drug combination and drug repurposing are two strategies that allow to find novel oncological therapies, in a faster and more economical process. In our previous studies, we developed a novel model of drug combination using antineoplastic and different repurposed drugs. We demonstrated the combinations of doxorubicin (DOX) + artesunate, DOX + chloroquine, paclitaxel (PTX) + fluoxetine, PTX + fluphenazine, and PTX + benztropine induce significant cytotoxicity in Michigan Cancer Foundation-7 (MCF-7) breast cancer cells. Furthermore, it was found that 5-FU + thioridazine and 5-fluorouracil (5-FU) + sertraline can synergistically induce a reduction in the viability of human colorectal adenocarcinoma cell line (HT-29). In this study, we aim to (1) evaluate the biosafety profile of these drug combinations for non-tumoral cells and (2) determine their mechanism of action in cancer cells. To do so, human fetal lung fibroblast cells (MRC-5) fibroblast cells were incubated for 48 h with all drugs, alone and in combination in concentrations of 0.25, 0.5, 1, 2, and 4 times their half-maximal inhibitory concentration (IC50). Cell morphology and viability were evaluated. Next, we designed and constructed a cell microarray to perform immunohistochemistry studies for the evaluation of palmitoyl-protein thioesterase 1 (PPT1), Ki67, cleaved-poly (ADP-ribose) polymerase (cleaved-PARP), multidrug resistance-associated protein 2 (MRP2), P-glycoprotein (P-gp), and nuclear factor-kappa-B (NF-kB) p65 expression. We demonstrate that these combinations are cytotoxic for cancer cells and safe for non-tumoral cells at lower concentrations. Furthermore, it is also demonstrated that PPT1 may have an important role in the mechanism of action of these combinations, as demonstrated by their ability to decrease PPT1 expression. These results support the use of antimalarial and central nervous system (CNS) drugs in combination regimens with chemotherapeutic agents; nevertheless, additional studies are recommended to further explore their complete mechanisms of action.

Characterization hiPSC-derived neural progenitor cells and neurons to investigate the role of NOS1AP isoforms in human neuron dendritogenesis.

Abnormal dendritic arbor development has been implicated in a number of neurodevelopmental disorders, such as autism and Rett syndrome, and the neuropsychiatric disorder schizophrenia. Postmortem brain samples from subjects with schizophrenia show elevated levels of NOS1AP in the dorsolateral prefrontal cortex, a region of the brain associated with cognitive function. We previously reported that the long isoform of NOS1AP (NOS1AP-L), but not the short isoform (NOS1AP-S), negatively regulates dendrite branching in rat hippocampal neurons. To investigate the role that NOS1AP isoforms play in human dendritic arbor development, we adapted methods to generate human neural progenitor cells and neurons using induced pluripotent stem cell (iPSC) technology. We found that increased protein levels of either NOS1AP-L or NOS1AP-S decrease dendrite branching in human neurons at the developmental time point when primary and secondary branching actively occurs. Next, we tested whether pharmacological agents can decrease the expression of NOS1AP isoforms. Treatment of human iPSC-derived neurons with d-serine, but not clozapine, haloperidol, fluphenazine, or GLYX-13, results in a reduction in endogenous NOS1AP-L, but not NOS1AP-S, protein expression; however, d-serine treatment does not reverse decreases in dendrite number mediated by overexpression of NOS1AP isoforms. In summary, we demonstrate how an in vitro model of human neuronal development can help in understanding the etiology of schizophrenia and can also be used as a platform to screen drugs for patients.

Antiviral activity of chlorpromazine, fluphenazine, perphenazine, prochlorperazine, and thioridazine towards RNA-viruses. A review.

In 2020 the whole world focused on antivirus drugs towards SARS-CoV-2. Most of the researchers focused on drugs used in other viral infections or malaria. We have not seen such mobilization towards one topic in this century. The whole situation makes clear that progress needs to be made in antiviral drug development. The first step to do it is to characterize the potential antiviral activity of new or already existed drugs on the market. Phenothiazines are antipsychotic agents used previously as antiseptics, anthelminthics, and antimalarials. Up to date, they are tested for a number of other disorders including the broad spectrum of viruses. The goal of this paper was to summarize the current literature on activity toward RNA-viruses of such drugs like chlorpromazine, fluphenazine, perphenazine, prochlorperazine, and thioridazine. We identified 49 papers, where the use of the phenothiazines for 23 viruses from different families were tested. Chlorpromazine, fluphenazine, perphenazine, prochlorperazine, and thioridazine possess anti-viral activity towards different types of viruses. These drugs inhibit clathrin-dependent endocytosis, cell-cell fusion, infection, replication of the virus, decrease viral invasion as well as suppress entry into the host cells. Additionally, since the drugs display activity at nontoxic concentrations they have therapeutic potential for some viruses, still, further research on animal and human subjects are needed in this field to verify cell base research.

Based on Principles and Insights of COVID-19 Epidemiology, Genome Sequencing, and Pathogenesis: Retrospective Analysis of Sinigrin and ProlixinRX (Fluphenazine) Provides Off-Label Drug Candidates.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative pathogen of pandemic coronavirus disease 2019 (COVID-19). So far, no approved therapy has been developed to halt the spread of the pathogen, and unfortunately, the strategies for developing a new therapy will require a long time and very extensive resources. Therefore, drug repurposing has emerged as an ideal strategy toward a smart, versatile, quick way to confine the lethal disease. In this endeavor, natural products have been an untapped source for new drugs. This review represents the confederated experience of multidisciplinary researchers of 99 articles using several databases: Google Scholar, Science Direct, MEDLINE, Web of Science, Scopus, and PubMed. To establish the hypothesis, a Bayesian perspective of a systematic review was used to outline evidence synthesis. Our docking documentation of 69 compounds and future research agenda assumptions were directed toward finding an effective and economic anti-COVID-19 treatment from natural products. Glucosinolate, flavones, and sulfated nitrogenous compounds demonstrate direct anti-SARS-CoV-2 activity through inhibition protease enzymes and may be considered potential candidates against coronavirus. These findings could be a starting point to initiate an integrative study that may encompass interested scientists and research institutes to test the hypothesis in vitro, in vivo, and in clinics after satisfying all ethical requirements.

In vitro and in vivo identification of clinically approved drugs that modify ACE2 expression.

The COVID-19 pandemic caused by SARS-CoV-2 has is a global health challenge. Angiotensin-converting enzyme 2 (ACE2) is the host receptor for SARS-CoV-2 entry. Recent studies have suggested that patients with hypertension and diabetes treated with ACE inhibitors (ACEIs) or angiotensin receptor blockers have a higher risk of COVID-19 infection as these drugs could upregulate ACE2, motivating the study of ACE2 modulation by drugs in current clinical use. Here, we mined published datasets to determine the effects of hundreds of clinically approved drugs on ACE2 expression. We find that ACEIs are enriched for ACE2-upregulating drugs, while antineoplastic agents are enriched for ACE2-downregulating drugs. Vorinostat and isotretinoin are the top ACE2 up/downregulators, respectively, in cell lines. Dexamethasone, a corticosteroid used in treating severe acute respiratory syndrome and COVID-19, significantly upregulates ACE2 both in vitro and in vivo. Further top ACE2 regulators in vivo or in primary cells include erlotinib and bleomycin in the lung and vancomycin, cisplatin, and probenecid in the kidney. Our study provides leads for future work studying ACE2 expression modulators.

Agonist-mediated switching of ion selectivity in TPC2 differentially promotes lysosomal function.

Ion selectivity is a defining feature of a given ion channel and is considered immutable. Here we show that ion selectivity of the lysosomal ion channel TPC2, which is hotly debated (Calcraft et al., 2009; Guo et al., 2017; Jha et al., 2014; Ruas et al., 2015; Wang et al., 2012), depends on the activating ligand. A high-throughput screen identified two structurally distinct TPC2 agonists. One of these evoked robust Ca2+-signals and non-selective cation currents, the other weaker Ca2+-signals and Na+-selective currents. These properties were mirrored by the Ca2+-mobilizing messenger, NAADP and the phosphoinositide, PI(3,5)P2, respectively. Agonist action was differentially inhibited by mutation of a single TPC2 residue and coupled to opposing changes in lysosomal pH and exocytosis. Our findings resolve conflicting reports on the permeability and gating properties of TPC2 and they establish a new paradigm whereby a single ion channel mediates distinct, functionally-relevant ionic signatures on demand.

Impact of calmodulin inhibition by fluphenazine on susceptibility, biofilm formation and pathogenicity of caspofungin-resistant Candida glabrata.

BACKGROUND: In recent decades, Candida glabrata has emerged as a frequent cause of life-threatening fungal infection. In C. glabrata, echinocandin resistance is associated with mutations in FKS1/FKS2 (ß-1,3-glucan synthase). The calmodulin/calcineurin pathway is implicated in response to antifungal stress and calcineurin gene disruption specifically reverses Fks2-mediated resistance of clinical isolates. OBJECTIVES: We evaluated the impact of calmodulin inhibition by fluphenazine in two caspofungin-resistant C. glabrata isolates. METHODS: C. glabrata isolates were identified by ITS1/ITS4 (where ITS stands for internal transcribed spacer) sequencing and the echinocandin target FKS1/FKS2 genes were sequenced. Susceptibility testing of caspofungin in the presence of fluphenazine was performed by a modified CLSI microbroth dilution method. The effect of the fluphenazine/caspofungin combination on heat stress (37°C or 40°C), oxidative stress (0.2 and 0.4 mM menadione) and biofilm formation (polyurethane catheter) was analysed. A Galleria mellonella model using blastospores (1 × 109 cfu/mL) was developed to evaluate the impact of this combination on larval survival. RESULTS: F659del was found in the FKS2 gene of both resistant strains. In these clinical isolates, fluphenazine increased susceptibility to caspofungin and reduced their thermotolerance. Furthermore, the fluphenazine/caspofungin combination significantly impaired biofilm formation in an in vitro polyurethane catheter model. All these features participated in the increasing survival of infected G. mellonella after combination treatment in comparison with caspofungin alone. CONCLUSIONS: In a repurposing strategy, our findings confirm that calmodulin could provide a relevant target in life-threatening fungal infectious diseases.

Dopamine D2 receptor influences eye development and function in Zebrafish. / El receptor de dopamina D2 influye en el desarrollo y la función del ojo en el pez cebra.

Dopamine is synthesized by tyrosine hydroxylase and is considered as a major catecholamine in the vertebrate retina, including zebrafish. However, little is known about the role of dopamine D2 receptor (DRD2) in retinal physiology. Therefore, to elucidate the role of DRD2 in the eye development and function in zebrafish, fish were exposed to fluphenazine, quinpirole, or combination of both. Subsequently, the eye size, optic nerve diameter (ONd), and visual background adaptation were evaluated. The results showed that fluphenazine (fluphenazine, DRD2 antagonist) decreased eye size and optic nerve diameter followed by disruption of visual function. The addition of Quinpirole (quinpirole, DRD2 agonist) reversed the effects caused by fluphenazine, implying that DRD2 is necessary for normal eye development and function in zebrafish. Considering the role of dopaminergic neurons in retinal development and function, dysfunction of dopaminergic neuron signaling pathways in the retina may cause visual abnormalities, particularly in the involvement of dopamine in regulating light response.

Influence of phenothiazine molecules on the interactions between positively charged poly-l-lysine and negatively charged DPPC/DPPG membranes.

Phenothiazines are very effective antipsychotic drugs, which also have anticancer and antimicrobial activities. Despite being used in human treatment, the molecular mechanism of the biological actions of these molecules is not yet understood in detail. The role of the interactions between phenothiazines and proteins or lipid membranes has been much discussed. Herein, fourier-transform infrared (FTIR) spectroscopic studies were used to investigate the effect of three phenothiazines: fluphenazine (FPh); chlorpromazine (ChP); and propionylpromazine (PP) on the structures of a positively charged poly-l-lysine (PLL) peptide, a negatively charged dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol (DPPC/DPPG) membrane, and on the mutual interactions between electrostatically associated PLL molecules and DPPC/DPPG membranes. Phenothiazine-induced alterations in the secondary structure of PLL, the conformational state (trans/gauche) of the hydrocarbon lipid chains, and the hydration of the DPPC/DPPG membrane interface were studied on the basis of amide I' vibrations, antisymmetric and symmetric stretching vibrations of the CH2 groups of the lipid hydrocarbon chains (νsCH2), and stretching vibrations of the lipid C=O groups (νC = O), respectively. It was shown that in the presence of negatively charged DPPC/DPPG membranes, the phenothiazines were able to modify the secondary structure of charged PLL molecules. Additionally, the effect of PLL on the structure of DPPC/DPPG membranes was also altered by the presence of the phenothiazine molecules.

Co-treatment With HIV Protease Inhibitor Nelfinavir Greatly Increases Late-phase Apoptosis of Drug-resistant KBV20C Cancer Cells Independently of P-Glycoprotein Inhibition.

BACKGROUND/AIM: The study focused on identifying the mechanisms or drugs that might sensitize resistant KBV20C human oral squamous carcinoma cells overexpressing P-glycoprotein (P-gp) to antimitotic drug treatment. MATERIALS AND METHODS: Five HIV protease inhibitors (atazanavir, nelfinavir, darunavir, lopinavir, and ritonavir) were tested to identify drugs that could be used at a relatively low dose for sensitizing antimitotic drug-resistant KBV20C cells. Fluorescence-activated cell sorting, annexin V analyses, and rhodamine uptake tests were performed to further investigate the mechanism of action. RESULTS: Co-treatment with nelfinavir or lopinavir had a high sensitizing effect on vincristine-treated KBV20C cells. Nelfinavir and lopinavir reduced cell viability, increased G2 phase arrest, and up-regulated apoptosis when used as a co-treatment with vincristine. We also demonstrated that eribulin co-treatment with nelfinavir and lopinavir similarly increased sensitization of KBV20C cells. Only lopinavir was found to have a high P-gp-inhibitory activity (similar to verapamil). Interestingly, nelfinavir had very low P-gp-inhibitory activity, suggesting that vincristine-nelfinavir sensitization is independent of the P-gp-inhibitory effect of nelfinavir. We also demonstrated this same combination mainly caused sensitization due to late apoptosis in P-gp-overexpressing drug-resistant KBV20C cells. CONCLUSION: Highly antimitotic drug-resistant KBV20C cells can be sensitized by co-treatment with the repositioned HIV protease inhibitors nelfinavir and lopinavir. In particular, the sensitizing effect of co-treatment with nelfinavir on antimitotic drug-resistant cancer cells was found to be strong and independent of P-gp-inhibitory activity. As P-gp inhibition can be toxic to normal cells, selecting nelfinavir may be safer for normal cells in patients with drug-resistant cancer.

Fluphenazine antagonizes with fluconazole but synergizes with amphotericin B in the treatment of candidiasis.

Candida albicans causes a high mortality rate in immunocompromised individuals, but the increased drug resistance challenges the current antifungal therapeutics. Fluphenazine (FPZ), a commonly used antipsychotic medication, can induce the expression of drug efflux pumps in C. albicans and, thus, may interfere with the therapeutic efficacy of antifungals, such as fluconazole (FLC) and amphotericin B (AmB). Here, we investigated the combined effects of FLC/FPZ and AmB/FPZ against C. albicans in vitro and in a systemic candidiasis mouse model. The antifungal activity of FLC was significantly reduced when supplemented with FPZ. The inhibitory effects of FLC on the expression of the Candida virulence-related genes ALS3 and HWP1 were antagonized by FPZ. However, FPZ enhanced the susceptibility of C. albicans to AmB and further downregulated the expression of ALS3 and HWP1 in a synergistic manner with AmB. FPZ also enhanced the gene expression of ERG11, a key gene of the ergosterol biosynthesis pathway that has been associated with the activities of both FLC and AmB. In our mammalian infection model, mice treated with FLC/FPZ showed notably poor living status and increased fungal burden in their kidneys and brains compared with those treated with FLC alone. Conversely, the combined application of AmB/FPZ significantly improved the survival rate, attenuated the weight loss and reduced the organ fungal burdens of the infected mice. These data suggest that FPZ antagonized the therapeutic efficacy of FLC but enhanced the antifungal activity of AmB in the treatment of candidiasis.

The neuroleptic drug fluphenazine induces a significant UVA-mediated cytotoxic effect on three human cancer cell lines through apoptosis.

The cytotoxic activity of fluphenazine (FPZ) in combination with UVA light was evaluated on three human tumor cell lines, HeLa, MSTO-211H and A431. The photobiological effect was determined following irradiation treatment in the presence of/or after the removal of incubated FPZ. Under both conditions, FPZ proved to be very effective in killing tumor cells, with GI50 values in the micromolar range. However, when FPZ was present during irradiation, the photocytotoxicity was at least two times higher than that after its removal suggesting the contribution of the drug both outside and inside the cells. The uptake of FPZ was very fast and, after only 15 minutes of incubation, the compound was accumulated inside lysosomes, as evidenced through fluorescence microscopy. FPZ distribution covered also the nucleus and the cytoplasm without significant plasma membrane association. After irradiation, the membrane of lysosomes in which FPZ was accumulated lost its integrity suggesting that the released lysosomal enzymes played an important role in cell death, and mitochondria were damaged as well, following apoptosis. Indeed, cytofluorimetric studies demonstrated that apoptosis was the main mechanism of cell death. Finally, an extremely high production of ROS was found, indicating a significant photodynamic mechanism involved in the photocytotoxic effect of FPZ. Taken together, our data show that FPZ following UVA irradiation behaves as an effective photoantiproliferative compound inducing apoptosis on various human tumor cells.

Increased lipid peroxidation, apoptosis and selective cytotoxicity in colon cancer cell line LoVo and its doxorubicin-resistant subline LoVo/Dx in the presence of newly synthesized phenothiazine derivatives.

Cancer cells often develop the resistance to pro-apoptotic signaling that makes them invulnerable to conventional treatment. Therapeutic strategies that make cancer cells enter the path of apoptosis are desirable due to the avoidance of inflammatory reaction that usually accompanies necrosis. In the present study phenothiazines (fluphenazine and four recently synthesized derivatives) were investigated in order to identify compounds with a potent anticancer activity. Since phenothiazines are known as multidrug resistance modulators the sensitive human colorectal adenocarcinoma cell line (LoVo) and its doxorubicin-resistant, ABCB1 overexpressing, subline (LoVo/Dx) have been employed as a model system. In studied cancer cells cytotoxic effect of the phenothiazine derivatives was accompanied by apoptosis and autophagy induction as well as by the increase of cellular lipid peroxidation and intracellular reactive oxygen species generation. Molecular modelling revealed that reactivity of phenothazines (manifested by their low energy gap) but not lipophilicity was positively correlated with their anticancer potency, pro-oxidant properties and apoptosis induction ability. Additionally, some of the studied compounds turned out to be more potent cytotoxic and pro-apoptotic agents in doxorubicin-resistant (LoVo/Dx) cells than in sensitive ones (LoVo). The hypothesis was assumed that studied phenothiazine derivatives induced apoptotic cell death by increasing the production of reactive oxygen species.

Osteocyte-Driven Downregulation of Snail Restrains Effects of Drd2 Inhibitors on Mammary Tumor Cells.

While bone is a frequent target of breast cancer-associated metastasis, little is known about the effects of tumor-bone interactions on the efficacy of tumor-suppressing agents. Here we examined the effect of two FDA-approved dopamine modulators, fluphenazine and trifluoperazine, on mammary tumor cells, osteoclasts, osteoblasts, and osteocytes. These agents suppressed proliferation and migration of mammary tumor cells chiefly by antagonizing dopamine receptor D2 and reduced bone resorption by downregulating nuclear factor of activated T cells, cytoplasmic 1 (Nfatc1). Three-dimensional spheroid formation assays revealed that tumor cells have high affinity to osteocytes and type I collagen, and interactions with osteocytes as well as administration of fluphenazine and trifluoperazine downregulated Snail and suppressed migratory behaviors. Unlike the inhibitory action of fluphenazine and trifluoperazine on tumor growth, tumor-osteocyte interactions stimulated tumor proliferation by upregulating NFκB and Akt. In the bone microenvironment, osteocytes downregulated Snail and acted as an attractant as well as a stimulant to mammary tumor cells. These results demonstrate that tumor-osteocyte interactions strengthen dopamine receptor-mediated suppression of tumor migration but weaken its inhibition of tumor proliferation in the osteocyte-rich bone microenvironment.Significance: These findings provide novel insight into the cellular cross-talk in the bone microevironment and the effects of dopamine modulators on mammary tumor cells and osteocytes. Cancer Res; 78(14); 3865-76. ©2018 AACR.

Modulating cancer cell survival by targeting intracellular cholesterol transport.

BACKGROUND: Demand for cholesterol is high in certain cancers making them potentially sensitive to therapeutic strategies targeting cellular cholesterol homoeostasis. A potential approach involves disruption of intracellular cholesterol transport, which occurs in Niemann-Pick disease as a result of acid sphingomyelinase (ASM) deficiency. Hence, a class of lysosomotropic compounds that were identified as functional ASM inhibitors (FIASMAs) might exhibit chemotherapeutic activity by disrupting cancer cell cholesterol homoeostasis. METHODS: Here, the chemotherapeutic utility of ASM inhibition was investigated. The effect of FIASMAs on intracellular cholesterol levels, cholesterol homoeostasis, cellular endocytosis and signalling cascades were investigated. The in vivo efficacy of ASM inhibition was demonstrated using melanoma xenografts and a nanoparticle formulation was developed to overcome dose-limiting CNS-associated side effects of certain FIASMAs. RESULTS: Functional ASM inhibitors inhibited intracellular cholesterol transport leading to disruption of autophagic flux, cellular endocytosis and receptor tyrosine kinase signalling. Consequently, major oncogenic signalling cascades on which cancer cells were reliant for survival were inhibited. Two tested ASM inhibitors, perphenazine and fluphenazine that are also clinically used as antipsychotics, were effective in inhibiting xenografted tumour growth. Nanoliposomal encapsulation of the perphenazine enhanced its chemotherapeutic efficacy while decreasing CNS-associated side effects. CONCLUSIONS: This study suggests that disruption of intracellular cholesterol transport by targeting ASM could be utilised as a potential chemotherapeutic approach for treating cancer.

The PPII-to-α-helix transition of poly-l-lysine in methanol/water solvent mixtures accompanied by fibrillar self-aggregation: An influence of fluphenazine molecules.

Fourier-transform infrared, vibrational circular dichroism spectroscopy and transmission electron microscopy are used to follow the structural changes of pure and fluphenazine (FPh)-mixed poly-l-lysine (PLL) triggered by variations of the methanol to water ratio in solvent mixtures. FPh molecules are used as an effective psychotic drug but with a strong Parkinson's-related side effect. To answer the question whether FPh molecules can modify the fibril development, the PLL polypeptide was used as a model of α-helix- and PPII-rich fibrils. It was stated that the presence of FPh molecules did not inhibit the creation of both types of PLL fibrils with clustering features. The methanol-poor aqueous solutions promote the formation of extended polyproline II (PPII) helices; however, the methanol-rich aqueous solutions induce the development of α-helices of both pure and FPh-mixed PLL. Unpredicted and interesting features of PLL fibrillogenesis are evidenced by the formation of uncommon fibrillar aggregates, which are developed in methanol/water solvents from PLL molecules rich in either α-helix or PPII structures. Possibility of PLL molecules to form ß-sheet-, α-helix- and PPII-rich fibrils demonstrating that fibrillogenesis is a common phenomenon, and fibrillar aggregates can be based on all of the basic protein secondary structures.

Functional inhibition of acid sphingomyelinase by Fluphenazine triggers hypoxia-specific tumor cell death.

Owing to lagging or insufficient neo-angiogenesis, hypoxia is a feature of most solid tumors. Hypoxic tumor regions contribute to resistance against antiproliferative chemotherapeutics, radiotherapy and immunotherapy. Targeting cells in hypoxic tumor areas is therefore an important strategy for cancer treatment. Most approaches for targeting hypoxic cells focus on the inhibition of hypoxia adaption pathways but only a limited number of compounds with the potential to specifically target hypoxic tumor regions have been identified. By using tumor spheroids in hypoxic conditions as screening system, we identified a set of compounds, including the phenothiazine antipsychotic Fluphenazine, as hits with novel mode of action. Fluphenazine functionally inhibits acid sphingomyelinase and causes cellular sphingomyelin accumulation, which induces cancer cell death specifically in hypoxic tumor spheroids. Moreover, we found that functional inhibition of acid sphingomyelinase leads to overactivation of hypoxia stress-response pathways and that hypoxia-specific cell death is mediated by the stress-responsive transcription factor ATF4. Taken together, the here presented data suggest a novel, yet unexplored mechanism in which induction of sphingolipid stress leads to the overactivation of hypoxia stress-response pathways and thereby promotes their pro-apoptotic tumor-suppressor functions to specifically kill cells in hypoxic tumor areas.

Diverse arrestin-recruiting and endocytic profiles of tricyclic antipsychotics acting as direct α2A adrenergic receptor ligands.

The therapeutic mechanism of action underlying many psychopharmacological agents remains poorly understood, due largely to the extreme molecular promiscuity exhibited by these agents with respect to potential central nervous system targets. Agents of the tricyclic chemical class, including both antidepressants and antipsychotics, exhibit a particularly high degree of molecular promiscuity; therefore, any clarification of how these agents interact with specific central nervous system targets is of great potential significance to the field. Here, we present evidence demonstrating that tricyclic antipsychotics appear to segregate into three distinct groups based upon their molecular interactions with the centrally-important α2A adrenergic receptor (AR). Specifically, while the α2AAR binds all antipsychotics tested with similar affinities, and none of the agents are able to induce classical heterotrimeric G protein-mediated α2AAR signaling, significant differences are observed with respect to arrestin3 recruitment and receptor endocytosis. All antipsychotics tested induce arrestin3 recruitment to the α2AAR, but with differing strengths. Both chlorpromazine and clozapine drive significant α2AAR endocytosis, but via differing clathrin-dependent and lipid raft-dependent pathways, while fluphenazine does not drive a robust response. Intriguingly, in silico molecular modeling suggests that each of the three exhibits unique characteristics in interacting with the α2AAR ligand-binding pocket. In addition to establishing these three antipsychotics as novel arrestin-biased ligands at the α2AAR, our findings provide key insights into the molecular actions of these clinically-important agents.