Trifluoperazine,a calmodulin antagonist,inhibits muscle cell fusion

We investigated the effect of trifluoperazine (TFP), a calmodulin antagonist, on the fusion of chick skeletal myoblasts in culture. TFP was found to inhibit myoblast fusion. This effect occurs at concentrations that have been reported to inhibit Ca2+-calmodulin in vitro, and is reversed upon removal of TFP. In addition, other calmodulin antagonists, including chlorpromazine, N-(6-aminohexyl)-5- chloro-1-naphthalene-sulfonamide (W7), and N-(6-aminohexyl)-1- naphthalene-sulfonamide (W5), inhibit fusion at doses that correspond closely to the antagonistic effects of these drugs on calmodulin. The expression of surface acetylcholine receptor, a characteristic aspect of muscle differentiation, is not impaired in TFP-arrested myoblasts. Myoblasts inhibited from fusion by 10 microM TFP display impaired alignment. In the presence of the Ca2+ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, the fusion block by 10 microM TFP is partially reversed and myoblast alignment is restored. The presence and distribution of calmodulin in both prefusional myoblasts and fused muscle cells was established by immunofluorescence. We observed an apparent redistribution of calmodulin staining that is temporally correlated with the onset of myoblast fusion. Our findings suggest a possible role for calmodulin in the regulation of myoblast fusion.     

A role for intracellular calcium and calmodulin in the release of triiodothyronine from human thyroid-cell monolayer cultures

Human thyroid cells in monolayer responded to acute stimulation by TSH with an increase in the secretion of T₃. This process appeared to be dependent on a rise in the cytosolic calcium concentration since the antagonist of intraceliular calcium mobilization, TMB-8, was found to inhibit the release of T₃ in response to TSH. The importance of intracellular calcium was further shown using the agent veratridine which increases the free calcium level within cells; veratridine potentiated the stimulation of T₃ secretion by TSH and itself stimulated the release of T₃ to a level higher than that seen in the presence of TSH alone. The calcium ionophore A23197 produced a biphasic effect on T₃ secretion from human thyroid monolayers; at low concentrations, A23187 caused a decrease in both unstimulated and TSH-stimulated T₃ secretion but above a concentration of 1 M, T₃ secretion was increased. The calmodulin antagonist W7 was found to inhibit T₃ release in response to TSH, indicating a role for calmodulin in mediating the effects of intracellular calcium on T₃ secretion.     

Paclitaxel probably enhances cytotoxicity of natural killer cells against breast carcinoma cells by increasing perforin production

Paclitaxel, a semisynthetic taxane, is one of the most active chemotherapeutic agents for the treatment of patients with breast cancer. We focused on the effect of paclitaxel on the cytotoxicity of natural killer (NK) cells. NK cells were purified by negative selection with magnetic beads from peripheral blood mononuclear cells of healthy volunteers. A human breast carcinoma cell line BT-474 and an NK cell–sensitive erythroleukemia cell line K562 were used as targets. Cytotoxicity of NK cells was determined by ⁵¹Cr-release assay with labeled target cells. Paclitaxel (1–100 nM) did not affect cellular viability, and significantly enhanced cytotoxicity of NK cells in a dose-dependent manner. Although paclitaxel did not affect Fas-ligand expression of NK cells, paclitaxel induced mRNA and protein production of perforin, an effector molecule in NK cell–mediated cytotoxicity. Concanamycin A, a potent inhibitor of the perforin-mediated cytotoxic pathway, inhibited paclitaxel-dependent NK cell–mediated cytotoxicity. Furthermore, paclitaxel induced activation of nuclear factor B (NF-B) in NK cells. NF-B inhibitor pyrrolidine dithiocarbamate significantly suppressed both paclitaxel-induced perforin expression and NK cell cytotoxicity. Our results show for the first time that paclitaxel enhances in vitro cytotoxicity of human NK cells. Moreover, our results suggest a significant association between enhanced NK cell cytotoxicity, increased perforin production, and NF-B activation.     

Inhibition of growth of C6 astrocytoma cells by inhibitors of calmodulin

We evaluated the effect of several classes of calmodulin inhibitors on the activity of calmodulin prepared from C6 astrocytoma cells and studied the activity of these drugs as inhibitors of the growth of C6 cells in tissue culture. There was a good correlation between the activity of the drugs as inhibitors of calmodulin and their activity as inhibitors of cell growth. The most potent compounds were calmidazolium and melittin as compared to the phenothiazines, trifluoperazine, chlorpromazine, chlorpromazine-sulfoxide or the diphenylbutylpiperidine, pimozide. The mechanism by which the inhibition of calmodulin leads to the death of cells could not be attributed entirely to inhibition of the calmodulin-sensitive cyclic nucleotide phosphodiesterase. Calmodulin is a heat stable, calcium-binding protein involved in numerous biological processes. Recent evidence indicates that calcium and calmodulin may be important for cellular proliferation. For example, this protein changes in concentration during the cell cycle; is involved in the disassembly of the mitotic apparatus; is increased in concentration in rapidly growing hepatomas and in transformed fibroblasts. Weiss and co-workers demonstrated that phenothiazines and structurally similar drugs are capable of binding to and inhibiting the activity of calmodulin. It has been recently observed that certain drugs that inhibit the activity of calmodulin also inhibit the growth of malignant cells in vitro and in vivo. In these studies, however, there was no direct correlation of the effect of the drugs on the calmodulin from the cell type under investigation with cytotoxicity. To learn more about the relationship between a drug's ability to inhibit calmodulin and its antiproliferative activity, we correlated the effect of drugs on the activity of calmodulin prepared from the C6 astrocytoma cell line with their effect on cellular proliferation. Since many inhibitors of calmodulin readily cross the blood-brain barrier and since no acceptable treatment for malignancies of the central nervous system exist, we chose this cell line as a model for elucidating the potential antineoplastic effects of calmodulin inhibitors.     

The role of apoptosis in antibody-dependent cellular cytotoxicity

Apoptosis in three lymphoma cell lines has been studied following cytotoxicity induced in vitro by normal human blood lymphocytes utilizing either natural killer (NK) or antibody-dependent cellular cytotoxic (ADCC) mechanisms. Guinea-pig L₂C leukaemic lymphocytes, but not the human cell lines Daudi and Jurkat, revealed a degree of time- and temperature-dependent apoptotic death upon simple culture in vitro. NK cytotoxicity at low effector: target ratios (E: T) induced both release of⁵¹Cr and apoptosis. However NK cytotoxicity at higher E : T, and ADCC at all E : T, increased the level of⁵¹Cr release while reducing the level of apoptosis. The findings were consistent with the apoptotic process being cut short by intervention of necrotic death. The same characteristics accompanied ADCC whether the effectors were recruited by Fc regions of antibody coating the targets, or by bispecific antibodies attaching one arm to the targets and the other to Fc receptors type III on effectors. This finding, and the high level of cytotoxicity elicited by the bispecific method, confirm the belief that NK cells, in addition to exerting NK cytotoxicity, represent the principal effectors for ADCC among blood mononuclear cells. Our results suggest that NK cells have both apoptotic and necrotic mechanisms available for killing their targets, but use only the latter for ADCC.     

Identification of calmodulin-like activity in term human amnion: effect of calmodulin inhibitors on prostaglandin biosynthesis

Human amnion prostaglandin E2 (PGE2) synthesis increases with the onset of labour, and this synthesis is Ca2+-dependent. To understand better the mechanism of Ca2+-stimulated PGE2 biosynthesis, studies were performed to identify the presence of the intracellular Ca2+-mediator, calmodulin, in human amnion and to examine its role in PGE2 synthesis. Calmodulin-like activity was identified by the ability of the microsomal and cytosolic fractions of the 105,000g centrifugation of amnion homogenate to stimulate cyclic AMP-dependent phosphodiesterase activity. Cytosolic fractions consistently stimulated phosphodiesterase activity more than microsomal fractions (P less than 0.001) in paired samples from term human amnions. This activity was calcium-dependent. The cytosolic and microsomal factors increased the Vmax but not the Km of phosphodiesterase. There were no differences in these parameters with the onset of labour. The distribution of calmodulin-like activity between microsomes and cytosol was similar to the distribution of calmodulin mass as determined by radioimmunoassay. Three structurally different inhibitors of calmodulin activity, calmidazolium, trifluoperazine and W7, were tested for their ability to inhibit cytosolic factor-stimulated phosphodiesterase activity and to inhibit PGE2 output from dispersed amnion cells obtained before the onset of labour at term (cesarean section cells) or after spontaneous labour and vaginal delivery (spontaneous labour cells). The 50% inhibitory concentrations of the calmodulin antagonists in the phosphodiesterase assay were: trifluoperazine (6.7 microM), calmidazolium (0.11 microM), and W7 (24 microM). Trifluoperazine inhibited both basal and calcium ionophore (A23187)-stimulated PGE2 output from cesarean section cells and spontaneous labour amnion cells. Calmidazolium inhibited basal PGE2 output in cesarean section cells and spontaneous labour cells, but had no effect on A23187-stimulated output. W7 inhibited only the ionophore-stimulated PGE2 output in cesarean section amnion cells. The rank order of inhibition of both phosphodiesterase activation and basal PGE2 output was: calmidazolium greater than trifluoperazine greater than W7. These results suggest that human amnion contains calmodulin and that its distribution, concentration and activity remain unchanged with the onset of labour. The data suggest, although not conclusively, that calmodulin may, in part, play a role in amnion cell PGE2 production. Further investigation of calmodulin effects upon specific enzymes in the PGE2 synthetic pathway will be necessary to elucidate a role for calmodulin in PGE2 production.     

Natural killer cell cytotoxicity: role of calmodulin

The phenothiazine derivatives, fluphenazine and trifluoperazine which are known to bind to calmodulin and to inhibit its activity, abrogate the development of both spontaneous and interferon-enhanced cytotoxicity of mouse splenic lymphocytes enriched for NK cell activity. Phenothiazines also inhibit the rapid increase in cyclic GMP levels in interferon-treated splenic lymphocytes. Furthermore, treatment of mouse splenic lymphocytes with electrophoretically pure interferon, alpha/beta caused a marked decrease in the level of calmodulin within 1 to 4 hours. These results provide evidence that calmodulin may play a role in the development of NK cell cytotoxicity and that the effect of interferon on calmodulin may constitute part of the molecular mechanism of interferon action.     

Studies on the involvement of calcium and calmodulin in the action of growth-hormone-releasing factor

A possible role for Ca 2⁺ and calmodulin in the action of growth-hormone-releasing factor (GHRF) was investigated . Low extracellular Ca²⁺ (<100 M), methoxyverapamil, flunarizine, cinnarizine, and Co²⁺ decreased both basal and GHRF-stimulated growth-hormone secretion, but did not totally inhibit GHRF-stimulation secretion. A calmodulin antagonist, W7, abolished GHRF-stimulated GH secretion, with no effect on basal secretion. It is suggested that GHRF may act primarily by elevating cellular cyclic AMP, which may then modulate calcium mobilization or flux; the increased intracellular Ca²⁺ concentrations may then activate calmodulin.     

Compounds containing 2-substituted imidazole ring for treatment against human African trypanosomiasis

A series of compounds containing 2-substituted imidazoles has been synthesized from imidazole and tested for its biological activity against human African trypanosomiasis (HAT). The 2-substituted 5-nitroimidazoles such as fexinidazole (7a) and 1-[4-(1-methyl-5-nitro-1H-imidazol-2-ylmethoxy)-pyridin-2-yl-piperazine (9e) exhibited potent activity against T. brucei in vitro with low cytotoxicity and good solubility. The presence of the NO₂ group at the 5-position of the imidazole ring in 2-substituted imidazoles is the crucial factor to inhibit T. brucei.     

Protein kinase C inhibition by calmodulin and its fragments

Inhibition of protein kinase C (PKC) by calmodulin is investigated and we describe the localization of inhibitory sequences within the calmodulin molecule. We present evidence that calmodulin inhibits PKC through an inhibition of the activation of PKC associated with lipid membranes: Binding of PKC to lipid vesicles is not affected, but activation is abolished. The potent calmodulin antagonist R24571 (calmidazol) did not affect the inhibition of PKC by calmodulin at concentrations up to 10^–5 M. Two tryptic fragments of calmodulin were isolated which inhibited PKC. They were only slightly less potent than intact calmodulin with an IC₅₀ of 6 µ M compared to 1 µ M of intact calmodulin. They were identified as Ser³⁸-Arg⁷⁴ and His¹⁰⁷-Lys¹⁴⁸. Each of the inhibiting fragments contains an intact Ca²⁺-binding domain with complete helix-loop-helix structure (EF hand). Other calmodulin peptides showed only weak inhibitory activity. Both fragments did not stimulate cAMP phosphodiesterase even at concentrations 100-fold higher than the calmodulin concentration needed for maximal stimulation. None of the fragments acted as a calmodulin antagonist.     

Synthesis, structure-activity relationship of novel substituted 4H-chromen-1,2,3,4-tetrahydropyrimidine-5-carboxylates as potential anti-mycobacterial and anticancer agents

Series of 4H-chromen-1,2,3,4-tetrahydropyrimidine-5-carboxylate derivatives 7a-7zb, 8a-8d and 9a-9d were synthesized and screened for their in vitro anti-mycobacterial activity against Mycobacterium tuberculosis H₃₇Rv (MTB) and cytotoxicity against three human cancer cell lines including A549, SK-N-SH and HeLa. The results indicate that six compounds are more potent and 7za is most effective anti-mycobacterial derivative compared to the standard drugs Ethambutol and Ciprofloxacin. However, 12 compounds exhibited cytotoxicity against human neuroblastoma cell line; amongst them the compound 7v is most effective compared to the standard drug Doxorubicin. This is the first report assigning in vitro anti-mycobacterial, anticancer and structure-activity relationship for this new class of 4H-chromen-1,2,3,4-tetrahydropyrimidine-5-carboxylates.     

Is calmodulin inhibition involved in shape transformations induced by amphiphiles in erythrocytes?

Potent antihaemolytic and shape transforming amphiphilic compounds were studied for their ability to inhibit calmodulin-activated phosphodiesterase activity. Some echinocytogenic and stomatocytogenic amphiphiles were potent calmodulin inhibitors. The most potent echinocytogenic and stomatocytogenic amphiphiles, however, had no or only weak inhibitory effect. Our results show that there is no causal relationship between the ability of amphiphiles to induce antihaemolysis or shape transformations in erythrocytes and their ability to inhibit calmodulin-activated phosphodiesterase activity, and it is suggested that calmodulin is not involved in shape transformations induced by amphiphiles.     

Synthesis of a novel series of L-isoserine derivatives as aminopeptidase N inhibitors

A series of novel L-isoserine derivatives were synthesised and evaluated for their ability to inhibit aminopeptidase N (APN)/CD13. In our preliminary biological results, some of these compounds possessed a potent inhibitory activity against the APN. Within this series, compound 14b not only showed similar enzyme inhibition (IC₅₀ of 12.2?μM) compared with the positive control bestatin (half maximal inhibitory concentration (IC₅₀) of 7.3?μM), but also had a potent antiproliferative activity against human cancer cell lines cells.     

New inhibitors of tyrosyl-DNA phosphodiesterase I (Tdp 1) combining 7-hydroxycoumarin and monoterpenoid moieties

A number of derivatives of 7-hydroxycoumarins containing aromatic or monoterpene substituents at hydroxy-group were synthesized based on a hit compound from a virtual screen. The ability of these compounds to inhibit tyrosyl-DNA phosphodiesterase I (Tdp 1), important target for anti-cancer therapy, was studied for the first time. It was found that the 7-hydroxycoumarin derivatives with monoterpene pinene moiety are effective inhibitors of Tdp 1 with the most active derivative (+)-25c with IC₅₀ value of 0.675 μM. This compound has low cytotoxicity (CC₅₀ >100 μM) when tested against human cancer cells which is crucial for presupposed application in combination with clinically established anticancer drugs. The ability of the new compounds to enhance the cytotoxicity of camptothecin, an established topoisomerase 1 poison, was demonstrated.     

Daurisoline derivatives inhibit the ability of calmodulin to stimulate cyclic nucleotide phosphodiesterase activity

Daurisoline alkaloid derivatives were found to be potent calmodulin (CaM) antagonists. The ability of daurisoline derivatives to attenuate the stimulatory effect on calmodulin activated cyclic nucleotides phosphodiesterase (CaM-PDE) was studied. These compounds did not inhibit the basal activity of this enzyme. The hydrophobicity of these compounds was related to their inhibitory potency. It is suggested that such drugs bind directly to calmodulin in a Ca2(+)-dependent fashion, as indicated by their ability to change calmodulin fluorescence.     

Identification of calmodulin in the green alga Mougeotia and its possible function in chloroplast reorientational movement

A soluble protein was isolated from Mougeotia by chloropromazine-sepharose 4 B affinity chromatography. The protein matches the properties of calmodulin in terms of heat stability, Ca²⁺-dependent electrophoretic mobility in sodium-dodecyl-sulfate polyacrylamide gels, and its ability to activate cyclic nucleotide phosphodiesterase in a Ca²⁺-dependent manner. Phytochrome-mediated chloroplast reorientational movement in Mougeotia was inhibited by the calmodulin antagonist trifluoperazine, a hydrophobic compound, or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a hydrophilic compound; 50% inhibition (IC₅₀) of chloroplast movement is caused by 20–50 mol l^-1 trifluoperazine or 100 mol l^-1 W-7. The Ca²⁺-calmodulin may act as an intermediate in the chloroplast reorientational response in Mougeotia governed by phytochrome.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - SDS sodium dodecyl sulfate - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide     

Design,synthesis, biological evaluation,and molecular modeling studies of chalcone-rivastigmine hybrids as cholinesterase inhibitors

A series of novel chalcone-rivastigmine hybrids were designed, synthesized, and tested in vitro for their ability to inhibit human acetylcholinesterase and butyrylcholinesterase. Most of the target compounds showed hBChE selective activity in the micro- and submicromolar ranges. The most potent compound 3 exhibited comparable IC₅₀ to the commercially available drug (rivastigmine). To better understand their structure activity relationships (SAR) and mechanisms of enzyme-inhibitor interactions, kinetic and molecular modeling studies including molecular docking and molecular dynamics (MD) simulations were carried out. Furthermore, compound 3 blocks the formation of reactive oxygen species (ROS) in SH-SY5Y cells and shows the required druggability and low cytotoxicity, suggesting this hybrid is a promising multifunctional drug candidate for Alzheimer’s disease (AD) treatment.     

Synthesis,antitumor activity evaluation and mechanistic study of novel hederacolchiside A1 derivatives bearing an aryl triazole moiety

In an attempt to arrive at a more potent antitumor agent than the parent natural saponin hederacolchiside A₁, 23 hederacolchiside A₁ derivatives (4a-4w) were synthesized via Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition and screened in vitro for cytotoxicity against six human cancer cell lines. The structure-activity relationship of these compounds was elucidated, and the biological screening results showed that most of the compounds exhibited moderate to high levels of antitumor activities against the tested cell lines and some of them displayed more potent inhibitory activities compared with hederacolchiside A₁. Compound 4f showed a 2- to 7-fold more potent activity than hederacolchiside A₁. The mechanistic study of 4f revealed that this compound can induce cell apoptosis in HepG2 cells via mitochondrial-mediated intrinsic pathways.     

The CySF-L2 factor from dialysable human leucocyte extract activates natural killer cytotoxicity by induction of interferon γ

Summary The mechanism of natural killer (NK) cytotoxicity activation of human peripheral blood mononuclear cells (PBMC) by CySF-L2 was elucidated. CySF-L2 is a cytotoxicity-stimulating factor isolated from dialysable human leucocyte extract, which activates NK cytotoxicity against NK-sensitive and insensitive tumour cells (K562; Daudi; Raji; MOLT4) when preincubated with effector cells for 72 h. CySF-L2-mediated activation was synergistic to interleukin-2(IL-2)-mediated activation of NK cytotoxicity. Induction of interferon (IFN) release was the crucial step during CySF-L2-mediated NK cytotoxicity activation since enhancement of NK activity was completely blocked when anti-IFN antibodies were present during treatment of PBMC. Anti-IFN, anti-TNF (tumour necrosis factor ) anti-IL-1 and anti-IL-2 antibodies showed no blocking effect. Analysis of the supernatant culture medium after 72 h incubation of PBMC and their highly purified subpopulations demonstrated that CySF-L2 induced release of IFN from CD3⁺T cells and CD56⁺CD3^– NK cells and of TNF and prostaglandin E₂ from monocytes. CySF-L2 was also capable of activating NK cytotoxicity of highly purified (98%) CD56⁺CD3^– NK cells as well as of monocytes (94% pure). Cell cooperation studies connected with analysis of cytokine release and enhancement of NK cytotoxicity indicated that CySF-L2 might play an essential role in the up and down regulation of NK cytotoxicity by the cytokine network.     

Design,Synthesis and Molecular Docking Studies of Some Tetrahydropyrimidine Derivatives as Possible Fascin Inhibitors

Eight derivatives of tetrahydropyrimidine scaffold were designed and prepared as hybrid compounds possessing the structural features of both monastrol as an anticancer drug and nifedipine as a fascin blocking agent. All of the compounds were evaluated for their cytotoxic potency and the ability to inhibit 4T1 breast cancer cells migration. Then, they were investigated in silico for their ability to inhibit the fascin protein using molecular docking simulation. The most potent compound was 4d and the weakest one was 4a according to the in vitro cytotoxicity assay. The corresponding IC₅₀ values were 193.70 and 248.75 μm , respectively. The least cytotoxic compound ( 4a ) was one of the strongest ones in binding to the fascin binding site according to the molecular docking results. 4a and 4e inhibited the 4T1 cells migration better than other compounds. They were more potent than nifedipine in inhibiting the migration process. In silico studies proved 4h to be the most potent fascin inhibitor in terms of ΔG_bind although it was not inhibiting migration. The controversy between the in vitro and in silico results may cancel the theory of the involvement of the fascin inhibition in the migration inhibition. However, the considerable antimigratory effects of some of the synthesized compounds encourage performing further in vivo experiments to introduce novel tumor metastasis inhibitors.