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The Journal of General Physiology Jan 2019Despite extensive efforts spanning multiple decades, the development of highly effective Ca sensitizers for the heart remains an elusive goal. Existing Ca sensitizers...
Despite extensive efforts spanning multiple decades, the development of highly effective Ca sensitizers for the heart remains an elusive goal. Existing Ca sensitizers have other targets in addition to cardiac troponin (cTn), which can lead to adverse side effects, such as hypotension or arrhythmias. Thus, there is a need to design Ca-sensitizing drugs with higher affinity and selectivity for cTn. Previously, we determined that many compounds based on diphenylamine (DPA) were able to bind to a cTnC-cTnI chimera with moderate affinity (K ∼10-120 µM). Of these compounds, 3-chlorodiphenylamine (3-Cl-DPA) bound most tightly (K of 10 µM). Here, we investigate 3-Cl-DPA further and find that it increases the Ca sensitivity of force development in skinned cardiac muscle. Using NMR, we show that, like the known Ca sensitizers, trifluoperazine (TFP) and bepridil, 3-Cl-DPA is able to bind to the isolated N-terminal domain (N-domain) of cTnC (K of 6 µM). However, while the bulky molecules of TFP and bepridil stabilize the open state of the N-domain of cTnC, the small and flexible 3-Cl-DPA molecule is able to bind without stabilizing this open state. Thus, unlike TFP, which drastically slows the rate of Ca dissociation from the N-domain of isolated cTnC in a dose-dependent manner, 3-Cl-DPA has no effect on the rate of Ca dissociation. On the other hand, the affinity of 3-Cl-DPA for a cTnC-TnI chimera is at least an order of magnitude higher than that of TFP or bepridil, likely because 3-Cl-DPA is less disruptive of cTnI binding to cTnC. Therefore, 3-Cl-DPA has a bigger effect on the rate of Ca dissociation from the entire cTn complex than TFP and bepridil. Our data suggest that 3-Cl-DPA activates the cTn complex via a unique mechanism and could be a suitable scaffold for the development of novel treatments for systolic heart failure.
Topics: Animals; Bepridil; Calcium; Diphenylamine; Female; Heart; Humans; Myocardium; Rats; Rats, Wistar; Trifluoperazine; Troponin C; Troponin I
PubMed: 30442775
DOI: 10.1085/jgp.201812131 -
Journal of Experimental & Clinical... Sep 2017Resistance to adjuvant radiotherapy is a major cause of treatment failure in patients with glioblastoma (GBM). Autophagy inhibitors have been shown to enhance the...
BACKGROUND
Resistance to adjuvant radiotherapy is a major cause of treatment failure in patients with glioblastoma (GBM). Autophagy inhibitors have been shown to enhance the efficacy of radiotherapy for certain solid tumors. However, current inhibitors do not penetrate the blood-brain-barrier (BBB). Here, we assessed the radiosensitivity effects of the antipsychotic drug trifluoperazine (TFP) on GBM in vitro and in vivo.
METHODS
U251 and U87 GBM cell lines as well as GBM cells from a primary human biopsy (P3), were used in vitro and in vivo to evaluate the efficacy of TFP treatment. Viability and cytotoxicity was evaluated by CCK-8 and clonogenic formation assays. Molecular studies using immunohistochemistry, western blots, immunofluorescence and qPCR were used to gain mechanistic insight into the biological activity of TFP. Preclinical therapeutic efficacy was evaluated in orthotopic xenograft mouse models.
RESULTS
IC50 values of U251, U87 and P3 cells treated with TFP were 16, 15 and 15.5 μM, respectively. TFP increased the expression of LC3B-II and p62, indicating a potential disruption of autophagy flux. These results were further substantiated by a decreased Lysotracker Red uptake, indicating impaired acidification of the lysosomes. We show that TFP and radiation had an additive effect when combined. This effect was in part due to impaired TFP-induced homologous recombination. Mechanistically we show that down-regulation of cathepsin L might explain the radiosensitivity effect of TFP. Finally, combining TFP and radiation resulted in a significant antitumor effect in orthotopic GBM xenograft models.
CONCLUSIONS
This study provides a strong rationale for further clinical studies exploring the combination therapy of TFP and radiation to treat GBM patients.
Topics: Animals; Autophagy; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Glioblastoma; Homologous Recombination; Humans; Mice; Microtubule-Associated Proteins; RNA-Binding Proteins; Radiation Tolerance; Trifluoperazine; Xenograft Model Antitumor Assays
PubMed: 28870216
DOI: 10.1186/s13046-017-0588-z -
Oncotarget Jul 2016Triple-negative breast cancer (TNBC) is the most lethal form of breast cancer. Lacking effective therapeutic options hinders treatment of TNBC. Here, we show that...
Triple-negative breast cancer (TNBC) is the most lethal form of breast cancer. Lacking effective therapeutic options hinders treatment of TNBC. Here, we show that bepridil (BPD) and trifluoperazine (TFP), which are FDA-approved drugs for treatment of schizophrenia and angina respectively, inhibit Akt-pS473 phosphorylation and promote FOXO3 nuclear localization and activation in TNBC cells. BPD and TFP inhibit survival and proliferation in TNBC cells and suppress the growth of TNBC tumors, whereas silencing FOXO3 reduces the BPD- and TFP-mediated suppression of survival in TNBC cells. While BPD and TFP decrease the expression of oncogenic c-Myc, KLF5, and dopamine receptor DRD2 in TNBC cells, silencing FOXO3 diminishes BPD- and TFP-mediated repression of the expression of these proteins in TNBC cells. Since c-Myc, KLF5, and DRD2 have been suggested to increase cancer stem cell-like populations in various tumors, reducing these proteins in response to BPD and TFP suggests a novel FOXO3-dependent mechanism underlying BPD- and TFP-induced apoptosis in TNBC cells.
Topics: Animals; Apoptosis; Bepridil; Breast Neoplasms; Cell Nucleus; Dopamine; Female; Forkhead Box Protein O3; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Kruppel-Like Transcription Factors; MCF-7 Cells; Mice; Mice, Nude; Neoplastic Stem Cells; Phosphorylation; Proto-Oncogene Proteins c-myc; RNA, Small Interfering; Receptors, Dopamine D2; Trifluoperazine; Triple Negative Breast Neoplasms
PubMed: 27283899
DOI: 10.18632/oncotarget.9881 -
Cell Death & Disease Sep 2018Women with aggressive triple-negative breast cancer (TNBC) are at high risk of brain metastasis, which has no effective therapeutic option partially due to the poor...
Women with aggressive triple-negative breast cancer (TNBC) are at high risk of brain metastasis, which has no effective therapeutic option partially due to the poor penetration of drugs across the blood-brain barrier. Trifluoperazine (TFP) is an approved antipsychotic drug with good bioavailability in brain and had shown anticancer effect in several types of cancer. It drives us to investigate its activities to suppress TNBC, especially the brain metastasis. In this study, we chose three TNBC cell lines MDA-MB-468, MDA-MB-231, and 4T1 to assess its anticancer activities along with the possible mechanisms. In vitro, it induced G0/G1 cell cycle arrest via decreasing the expression of both cyclinD1/CDK4 and cyclinE/CDK2, and stimulated mitochondria-mediated apoptosis. In vivo, TFP suppressed the growth of subcutaneous xenograft tumor and brain metastasis without causing detectable side effects. Importantly, it prolonged the survival of mice bearing brain metastasis. Immunohistochemical analysis of Ki67 and cleaved caspase-3 indicated TFP could suppress the growth and induce apoptosis of cancer cells in vivo. Taken together, TFP might be a potential available drug for treating TNBC with brain metastasis, which urgently needs novel treatment options.
Topics: Animals; Antipsychotic Agents; Apoptosis; Brain; Brain Neoplasms; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Female; G1 Phase; Humans; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; Mice, Nude; Mice, SCID; Mitochondria; Resting Phase, Cell Cycle; Trifluoperazine; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays
PubMed: 30258182
DOI: 10.1038/s41419-018-1046-3 -
British Medical Journal Apr 1967
Topics: Adult; Female; Fluphenazine; Humans; Male; Movement Disorders; Perphenazine; Pregnancy; Trifluoperazine
PubMed: 6021008
DOI: 10.1136/bmj.2.5543.55-a -
Journal of Bacteriology May 2017FabG performs the NADPH-dependent reduction of β-keto acyl-acyl carrier protein substrates in the elongation cycle of fatty acid synthesis. We report the...
FabG performs the NADPH-dependent reduction of β-keto acyl-acyl carrier protein substrates in the elongation cycle of fatty acid synthesis. We report the characterization of a temperature-sensitive mutation (Δ) in that results from an in-frame 8-amino-acid residue deletion in the α6/α7 subdomain. This region forms part of one of the two dimerization interfaces of this tetrameric enzyme and is reported to undergo significant conformational changes upon cofactor binding, which define the entrance to the active-site cleft. The activity of the mutant enzyme is extremely thermolabile and is deficient in forming homodimers at nonpermissive temperatures with a corresponding decrease in fatty acid synthesis both and Surprisingly, the Δ strain reverts to temperature resistance at a rate reminiscent of that of a point mutant with intragenic pseudorevertants located either on the 2-fold axes of symmetry or at the mouth of the active-site cleft. The Δ mutation also confers resistance to the calmodulin inhibitor trifluoperazine and renders the enzyme extremely sensitive to Ca We also observed a significant alteration in the lipid A fatty acid composition of Δ strains but only in an background, probably due to alterations in the permeability of the outer membrane. These observations provide insights into the structural dynamics of FabG and hint at yet another point of regulation between fatty acid and lipid A biosynthesis. Membrane lipid homeostasis and its plasticity in a variety of environments are essential for bacterial survival. Since lipid biosynthesis in bacteria and plants is fundamentally distinct from that in animals, it is an ideal target for the development of antibacterial therapeutics. FabG, the subject of this study, catalyzes the first cofactor-dependent reduction in this pathway and is active only as a tetramer. This study examines the interactions responsible for tetramerization through the biochemical characterization of a novel temperature-sensitive mutation caused by a short deletion in an important helix-turn-helix motif. The mutant strain has altered phospholipid and lipid A compositions and is resistant to trifluoperazine, an inhibitor of mammalian calmodulin. Understanding its structural dynamics and its influence on lipid A synthesis also allows us to explore lipid homeostasis as a mechanism for antibiotic resistance.
Topics: Alcohol Oxidoreductases; Anti-Bacterial Agents; Calcium; Drug Resistance, Bacterial; Enzyme Stability; Escherichia coli; Lipid Metabolism; Protein Multimerization; Sequence Deletion; Suppression, Genetic; Temperature; Trifluoperazine
PubMed: 28264990
DOI: 10.1128/JB.00074-17 -
Pflugers Archiv : European Journal of... Aug 2009Trifluoperazine (TFP), a phenothiazine, is a commonly used antipsychotic drug whose therapeutic effects are attributed to its central anti-adrenergic and...
Trifluoperazine (TFP), a phenothiazine, is a commonly used antipsychotic drug whose therapeutic effects are attributed to its central anti-adrenergic and anti-dopaminergic actions. However, TFP is also a calmodulin (CaM) antagonist and alters the Ca(2+) binding properties of calsequestrin (CSQ). The CaM and CSQ proteins are known modulators of sarcoplasmic reticulum (SR) Ca(2+) release in ventricular myocytes. We explored TFP actions on cardiac SR Ca(2+) release in cells and single type-2 ryanodine receptor (RyR2) channel activity in bilayers. In intact and permeabilized ventricular myocytes, TFP produced an initial activation of RyR2-mediated SR Ca(2+) release and over time depleted SR Ca(2+) content. At the single channel level, TFP or nortryptiline (NRT; a tricyclic antidepressant also known to modify CSQ Ca(2+) binding) increased the open probability (Po) of CSQ-free channels with an EC(50) of 5.2 microM or 8.9 microM (respectively). This Po increase was due to elevated open event frequency at low drug concentrations while longer mean open events sustained Po at higher drug concentrations. Activation of RyR2 by TFP occurred in the presence or absence of CaM. TFP may also inhibit SR Ca uptake as well as increase RyR2 opening. Our results suggest TFP and NRT can alter RyR2 function by interacting with the channel protein directly, independent of its actions on CSQ or CaM. This direct action may contribute to the clinical adverse cardiac side effects associated with these drugs.
Topics: Animals; Calcium Channel Agonists; Calcium Signaling; Cats; Cells, Cultured; Dose-Response Relationship, Drug; Myocytes, Cardiac; Rabbits; Ryanodine Receptor Calcium Release Channel; Trifluoperazine; Ventricular Function, Left
PubMed: 19277699
DOI: 10.1007/s00424-009-0658-y -
The Journal of Biological Chemistry Nov 1993Calsequestrin is an intermediate affinity, high capacity Ca(2+)-binding protein found in the lumen of the sarcoplasmic reticulum of both skeletal and cardiac muscle...
Calsequestrin is an intermediate affinity, high capacity Ca(2+)-binding protein found in the lumen of the sarcoplasmic reticulum of both skeletal and cardiac muscle cells. Previous sequence analysis suggested that calsequestrin may contain a hydrophobic binding site for the drug trifluoperazine, a site shared by the calmodulin family and shown to play a role in calmodulin/calmodulin receptor interaction. Previous studies showed that, upon Ca2+ binding, calsequestrin undergoes a conformational change, burying the trifluoperazine-binding site, folding into a more compact structure that is trypsin-resistant, and increasing the negative ellipticity of the circular dichroism spectrum. In this study, the structural and functional roles of the trifluoperazine-binding site in the Ca(2+)-induced conformational change of calsequestrin are further studied using the calmodulin antagonists trifluoperazine and melittin. If trifluoperazine or melittin is added to calsequestrin prior to Ca2+ addition, then Ca(2+)-induced folding is inhibited as determined by the changes in circular dichroism spectra and protein sensitivity to trypsin digestion. If, however, Ca2+ is added prior to trifluoperazine or melittin, calsequestrin remains resistant to trypsin digestion, just as if the calmodulin antagonists are not present, suggesting that the conformational change is not affected. Aggregates of calsequestrin that exhibit high Ca2+ binding capacity have previously been shown to occur at high Ca2+ and calsequestrin concentrations. By preventing a prerequisite folding step, trifluoperazine or melittin also prevents the Ca(2+)-induced aggregation of calsequestrin, thus decreasing the maximal Ca2+ binding by calsequestrin. These data suggest that the trifluoperazine-binding site is critically involved in the Ca(2+)-induced intramolecular folding step required for the intermolecular interactions leading to high capacity Ca(2+)-binding by calsequestrin.
Topics: Amino Acid Sequence; Animals; Binding Sites; Calcium; Calsequestrin; Circular Dichroism; Melitten; Molecular Sequence Data; Muscles; Protein Binding; Protein Folding; Rabbits; Sarcoplasmic Reticulum; Trifluoperazine; Trypsin
PubMed: 8227022
DOI: No ID Found -
Scientific Reports Jan 2017Intrinsically disordered proteins (IDPs) are prevalent in eukaryotes, performing signaling and regulatory functions. Often associated with human diseases, they...
Intrinsically disordered proteins (IDPs) are prevalent in eukaryotes, performing signaling and regulatory functions. Often associated with human diseases, they constitute drug-development targets. NUPR1 is a multifunctional IDP, over-expressed and involved in pancreatic ductal adenocarcinoma (PDAC) development. By screening 1120 FDA-approved compounds, fifteen candidates were selected, and their interactions with NUPR1 were characterized by experimental and simulation techniques. The protein remained disordered upon binding to all fifteen candidates. These compounds were tested in PDAC-derived cell-based assays, and all induced cell-growth arrest and senescence, reduced cell migration, and decreased chemoresistance, mimicking NUPR1-deficiency. The most effective compound completely arrested tumor development in vivo on xenografted PDAC-derived cells in mice. Besides reporting the discovery of a compound targeting an intact IDP and specifically active against PDAC, our study proves the possibility to target the 'fuzzy' interface of a protein that remains disordered upon binding to its natural biological partners or to selected drugs.
Topics: Animals; Antineoplastic Agents; Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Ductal; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cellular Senescence; Drug Discovery; Drug Resistance, Neoplasm; Humans; Intrinsically Disordered Proteins; Mice; Mice, Nude; Molecular Targeted Therapy; Neoplasm Proteins; Pancreatic Neoplasms; Protein Binding; Trifluoperazine; Xenograft Model Antitumor Assays
PubMed: 28054562
DOI: 10.1038/srep39732 -
Microbial Cell Factories Jun 2015Getting access to authentic human drug metabolites is an important issue during the drug discovery and development process. Employing recombinant microorganisms as...
BACKGROUND
Getting access to authentic human drug metabolites is an important issue during the drug discovery and development process. Employing recombinant microorganisms as whole-cell biocatalysts constitutes an elegant alternative to organic synthesis to produce these compounds. The present work aimed for the generation of an efficient whole-cell catalyst based on the flavin monooxygenase isoform 2 (FMO2), which is part of the human phase I metabolism.
RESULTS
We show for the first time the functional expression of human FMO2 in E. coli. Truncations of the C-terminal membrane anchor region did not result in soluble FMO2 protein, but had a significant effect on levels of recombinant protein. The FMO2 biocatalysts were employed for substrate screening purposes, revealing trifluoperazine and propranolol as FMO2 substrates. Biomass cultivation on the 100 L scale afforded active catalyst for biotransformations on preparative scale. The whole-cell conversion of trifluoperazine resulted in perfectly selective oxidation to 48 mg (46% yield) of the corresponding N (1)-oxide with a purity >98%.
CONCLUSIONS
The generated FMO2 whole-cell catalysts are not only useful as screening tool for human metabolites of drug molecules but more importantly also for their chemo- and regioselective preparation on the multi-milligram scale.
Topics: Biocatalysis; Dinitrocresols; Escherichia coli; Gene Expression; Humans; Mixed Function Oxygenases; Pharmaceutical Preparations; Propranolol; Recombinant Proteins; Substrate Specificity; Trifluoperazine
PubMed: 26062974
DOI: 10.1186/s12934-015-0262-0