Ligand-based design identifies a potent NUPR1 inhibitor exerting anticancer activity via necroptosis.

Intrinsically disordered proteins (IDPs) are emerging as attractive drug targets by virtue of their prevalence in various diseases including cancer. Drug development targeting IDPs is challenging because they have dynamical structure features and conventional drug design is not applicable. NUPR1 is an IDP playing an important role in pancreatic cancer. We previously reported that Trifluoperazine (TFP), an antipsychotic agent, was capable of binding to NUPR1 and inhibiting tumors growth. Unfortunately, TFP showed strong central nervous system side-effects. In this work, we undertook a multidisciplinary approach to optimize TFP, based on the synergy of computer modeling, chemical synthesis, and a variety of biophysical, biochemical and biological evaluations. A family of TFP-derived compounds was produced and the most active one, named ZZW-115, showed a dose-dependent tumor regression with no neurological effects and induced cell death mainly by necroptosis. This study opens a new perspective for drug development against IDPs, demonstrating the possibility of successful ligand-based drug design for such challenging targets.

Repositioning of the antipsychotic trifluoperazine: Synthesis, biological evaluation and in silico study of trifluoperazine analogs as anti-glioblastoma agents.

Repositioning of the antipsychotic drug trifluoperazine for treatment of glioblastoma, an aggressive brain tumor, has been previously suggested. However, trifluoperazine did not increase the survival time in mice models of glioblastoma. In attempt to identify an effective trifluoperazine analog, fourteen compounds have been synthesized and biologically in vitro and in vivo assessed. Using MTT assay, compounds 3dc and 3dd elicited 4-5 times more potent inhibitory activity than trifluoperazine with IC50 = 2.3 and 2.2 µM against U87MG glioblastoma cells, as well as, IC50 = 2.2 and 2.1 µM against GBL28 human glioblastoma patient derived primary cells, respectively. Furthermore, they have shown a reasonable selectivity for glioblastoma cells over NSC normal neural cell. In vivo evaluation of analog 3dc confirmed its advantageous effect on reduction of tumor size and increasing the survival time in brain xenograft mouse model of glioblastoma. Molecular modeling simulation provided a reasonable explanation for the observed variation in the capability of the synthesized analogs to increase the intracellular Ca2+ levels. In summary, this study presents compound 3dc as a proposed new tool for the adjuvant chemotherapy of glioblastoma.

Identification of a Drug Targeting an Intrinsically Disordered Protein Involved in Pancreatic Adenocarcinoma.

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.

Integrins regulate neuronal neurotrophin gene expression through effects on voltage-sensitive calcium channels.

Integrin adhesion receptors regulate gene expression during growth and differentiation in various cell types. Recent work, implicating integrins in functional synaptic plasticity, suggest they may have similar activities in adult brain. The present study tested if integrins binding the arginine-glycine-aspartate (RGD) matrix sequence regulate neurotrophin and neurotrophin receptor gene expression in cultured hippocampal slices. The soluble RGD-containing peptide glycine-arginine-glycine-aspartate-serine-proline (GRGDSP) increased neurotrophin mRNA levels in transcript- and subfield-specific fashions. Integrin ligand effects were greatest for brain-derived neurotrophic factor transcripts I and II and barely detectable for transcript III. In accordance with increased nerve growth factor mRNA levels, GRGDSP increased c-fos expression as well. In contrast, growth-associated protein-43, amyloid precursor protein and fibroblast growth factor-1 mRNAs were not elevated. Ligand effects on brain-derived neurotrophic factor transcript II and c-fos mRNA did not depend on the integrity of the actin cytoskeleton, neuronal activity, or various signaling pathways but were blocked by L-type voltage-sensitive calcium-channel blockers. These results indicate that in mature hippocampal neurons integrin engagement regulates expression of a subset of growth-related genes at least in part through effects on calcium influx. Accordingly, these synaptic adhesion receptors may play the same role in maintaining an adult, differentiated state in brain as they do in other tissues and changes in integrin activation and/or engagement may contribute to dynamic changes in neurotrophin expression and to neuronal calcium signaling.

Calmodulin antagonists do not inhibit IK(Ca) channels of human erythrocytes.

Patch-clamp recordings were performed to study the effects of three calmodulin (CaM) antagonists on the gating of intermediate calcium-activated K(+) channels (IK(Ca)) of human erythrocytes. In the cell-attached configuration, both opening frequency and open probability of IK(Ca) channels were not significantly different in control cells and in those incubated with calmidazolium, trifluoperazine or W7. IK(Ca) channels in excised membrane patches, were normally activated by the calcium bathing the cytoplasmic side in the presence of CaM antagonists, at calcium concentrations ranging from 10(-7) to 10(-3) M. The activity of IK(Ca) channels, which had been previously up-modulated by an endogenous cAMP-dependent protein kinase, was not inhibited when perfused with CaM antagonists. The results presented in this study demonstrate that calmodulin antagonists do not inhibit the activity of native IK(Ca) channels of human erythrocytes. These data are in accordance with findings on the cloned IK(Ca) indicating that calmodulin is constitutively associated with these channels.

Polarized sphingolipid transport from the subapical compartment: evidence for distinct sphingolipid domains.

In polarized HepG2 cells, the sphingolipids glucosylceramide and sphingomyelin (SM), transported along the reverse transcytotic pathway, are sorted in subapical compartments (SACs), and subsequently targeted to either apical or basolateral plasma membrane domains, respectively. In the present study, evidence is provided that demonstrates that these sphingolipids constitute separate membrane domains at the luminal side of the SAC membrane. Furthermore, as revealed by the use of various modulators of membrane trafficking, such as calmodulin antagonists and dibutyryl-cAMP, it is shown that the fate of these separate sphingolipid domains is regulated by different signals, including those that govern cell polarity development. Thus under conditions that stimulate apical plasma membrane biogenesis, SM is rerouted from a SAC-to-basolateral to a SAC-to-apical pathway. The latter pathway represents the final leg in the transcytotic pathway, followed by the transcytotic pIgR-dIgA protein complex. Interestingly, this pathway is clearly different from the apical recycling pathway followed by glucosylceramide, further indicating that randomization of these pathways, which are both bound for the apical membrane, does not occur. The consequence of the potential coexistence of separate sphingolipid domains within the same compartment in terms of "raft" formation and apical targeting is discussed.

Trifluoperazine and its metal complexes inhibit the Moloney leukemia virus reverse transcriptase.

Reverse transcriptase plays an essential role in the early steps of the replicative cycle of retroviruses. Because of the resistance against nucleoside analogue inhibitors such as 3'-azido-2',3'-dideoxythymidine, the importance of the investigation of non-nucleoside analogue inhibitors is increasing. We have investigated the influence of trifluoperazine (TFP--a species of phenothiazines) and its newly prepared TFP-metal complexes (TFP-VO(IV), TFP-Cu(II), TFP-Ni(II), TFP-Pd(II), TFP-Sn(IV)). The compounds were tested on Moloney murine leukemia virus reverse transcriptase assay. The inhibitory effect of metal complexes was higher than that of TFP. TFP-VO(IV) showed higher effectiveness compared the added effect of parent tricyclic chemical and metal. Therefore we concluded that the improved biological action depends on the formation of metal complexes. This phenothiazine and its metal coordination complexes could become a new non-nucleoside analogue group of compounds inhibiting the retrovirus replication.

Potential anti-echinococcal activity of alkylaminoethers.

Trans 2-phenoxy cyclohexanol ethers (IA, IIA, IIIA, IVA, VA, and VIA), the cyclohexanol analog (IB) and one coumarinic compound (IC) were obtained and their activity against Echinococcus multilocularis metacestodes was studied and compared with that of trifluoperazine (TFP). All of these compounds are analogous to IA and belong to three classes. Class A comprises trans 2-phenoxycyclohexanol aminoethers whose alkylaminoether group varies; compound VIA bears one more methylene in its aminoether group than does compound IA. Class B consists of one compound exhibiting no phenoxy function. Class C comprises one coumarinic analog. In vitro assays were performed using metacestodes whose protoscoleces were attached to the germinal layer in open and in closed vesicles. Compounds IA and IIA exhibited the highest activity, but it was lower than that displayed by TFP under the same conditions. Compound IA was tested in an in vivo assay in jirds (50 mg/kg/daily beginning at 80 days p.i.); it produced results that were analogous to those obtained using TFP without inducing the neuroleptic effect associated with the latter. After 40-90 days' treatment, the percentage of diminution in the entire parasitic mass in the jirds that survived minimal treatment (71%) was about 41% as compared with that in untreated jirds. Histologic examination of the parasites in treated jirds revealed numerous dead protoscoleces and some parasitic dedifferentiated cells. This parasitic response may indicate that in alveolar echinococcosis, these drugs exhibit only a parasitostatic effect.

Receptor binding potencies of chlorpromazine, trifluoperazine, fluphenazine and their 10-N-substituted analogues.

Receptor interactions of three pairs of phenothiazine derivatives, namely chlorpromazine, trifluoperazine, fluphenazine and their dialkylaminoacyl analogues were investigated. The data indicate that neuroleptic derivatives are less effective at dopamine (D2), alpha 1-adrenoceptors (AR) and histamine receptors (H1). No differences in affinity to alpha 2-AR were observed. The chlorpromazine analogue was more effective at muscarinic receptors of both types.

Theoretical studies of the binding of trifluoperazine derivatives to site (82-93) of calmodulin: effect of lengthenings of the methylene linker chain on the binding affinity.

A theoretical study was performed of the comparative binding affinities to fragment (82-93) of calmodulin (CaM) of trifluoperazine (TFP) and three derivatives, in which the methylene chain linking the phenothiazine ring and the piperazinium group was lengthened by addition of one to three methylenes. The backbone of the oligopeptide was held in the alpha-helical conformation. The computations were performed with the SIBFA procedures (sum of interactions between fragments computed ab initio), which use empirical formulas based on ab initio self-consistent field computations. The interaction energy is the sum of the intermolecular phenothiazine derivative-oligopeptide interaction energy and of the separate intramolecular energy variations of the ligand, on the one hand, and of the oligopeptide, on the other hand, upon relaxing the conformations of side chains Glu 84, Glu 87, Phe 89 and Phe 92 due to complex formation. All three derivatives were found to display a higher binding affinity than did TFP itself, an optimal affinity being found for a four- and a five-methylene linker chain. In as much as fragment (82-93) of CaM is a plausible candidate receptor site for phenothiazines, these results imply that two such compounds should be endowed with a significantly greater anti-CaM activity than TFP itself.

Biochemical and ultrastructural investigation of the effect of Stelazine (trifluoperazine) on Hymenolepis diminuta (Cestoda).

The effects of the phenothiazine, Stelazine, on Hymenolepis diminuta were investigated. The cestode was incubated for 10 min at 37 degrees C with 1 mM trifluoperazine, in the presence and absence of Ca2+. Assay of brush border enzymes showed that drug treatment lowered the activities of alkaline phosphatase, Ca2+-ATP'ase, 5'-nucleotidase and type 1 phosphodiesterase. This occurred in parallel with a significant reduction in tegumental protein. Under these conditions gross changes in ultrastructural appearance and cellular organization were observed. There was a lack of ordered microtriches and the distal cytoplasm was absent. Glycogen granules were scattered throughout the cytoplasm within the subtegumental layer. The connective tissue also appeared to be in some disarray. The effects of Stelazine appeared to be dependent on time and were significantly increased when Ca2+ was included in the incubation medium. Incubation with the less hydrophobic phenothiazine trifluoperazine sulphoxide had minimal effect on the integrity of the cestode. The results reported here support the premise that certain phenothiazines may be considered as potential cestocidal agents.

Effect of phenothiazines on Hymenolepis diminuta with special reference to the brush border Ca2+-dependent ATPase.

Incubation of Hymenolepis diminuta with the calmodulin antagonist trifluoperazine causes lesions in the brush border of the cestode. Exposure to a phenothiazine of lower lipophilicity, trifluoperazine sulphoxide, had little effect. Characterisation of isolated brush border revealed two forms of Ca2+-ATPase which exhibited maximum activity at pH 5.5 and 7.5. Both forms were Ca2+-dependent but only the latter was influenced by calmodulin and trifluoperazine. It is suggested that the Ca2+-ATPase present in the tapeworm brush border may be the site of trifluoperazine toxicity.

Possible role of calmodulin in the control of histamine release from human basophil leukocytes.

We investigated the possible role of calmodulin (CaM) in the control of histamine release from human basophil leukocytes using several CaM antagonists. Trifluoperazine (TFP) (10(-6)-2 X 10(-5) M), pimozide (10(-6)-1.5 X 10(-5) M), chlorpromazine (CPZ) (10(-5)-10(-4) M) and promethazine (PMZ) (2 X 10(-5)-10(-4) M) inhibited in vitro histamine secretion from human basophils induced by several immunological (antigen, anti-IgE, and formyl-L-methionyl-L-leucyl-L-phenylalanine: f-met peptide) and nonimmunological (Ca2+ ionophore A23187 and the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate: TPA) stimuli. Trifluoperazine sulfoxide (TFP-S) and chlorpromazine sulfoxide (CPZ-S), which have very low affinity to CaM, had practically no inhibitory effect on histamine release from human basophils. The inhibitory effect of TFP could be made irreversible by irradiating the cells with UV light. A sulfonamide derivative, the compound N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) (2.5 X 10(-5)-2 X 10(-4) M), which selectively binds to CaM, inhibited the release of histamine from basophils. In contrast, the chloride deficient analogue, W-5, which interacts only weakly with CaM, had practically no inhibiting effect. The IC50 for enzyme release by a series of eight CaM antagonists was closely correlated (r = 0.91; p less than 0.001) with the CaM specific binding, supporting the concept that these agents act by binding to CaM and thereby inhibiting histamine release. TFP and W-7 inhibited histamine release in the absence and in the presence of increasing concentrations of extracellular Ca2+. These results emphasize the possible role of CaM in the control of histamine secretion from human basophils.

Development and application of a specific radioimmunoassay for trifluoperazine N4'-oxide to a kinetic study in humans.

A hapten derivative of prochlorperazine N4'-oxide, 7-(3-carboxy-propionyl)-prochlorperazine N4'-oxide, was synthesized and coupled to bovine serum albumin. Immunization of New Zealand White rabbits with this conjugate generated antibodies which were utilized to develop a radioimmunoassay (RIA) for the quantitation of trifluoperazine N4'-oxide (TFPNO). This assay enabled for the first time the quantitation of TFPNO in plasma, and 20 pg can be measured in a 200-microliter plasma sample with a coefficient of variation less than 5%. Statistically, indistinguishable results were obtained by the assay procedure with or without the selective extraction of TFPNO, and also in the presence or absence of an excess of trifluoperazine (TFP) and other major metabolites, such as trifluoperazine sulfoxide and 7-hydroxytrifluoperazine. This RIA and a previously reported RIA for TFP were used to directly determine plasma concentrations of TFP and TFPNO after administration of a single 5-mg p.o. dose of TFP to six healthy male volunteers. The mean +/- S.D. for the peak concentration, time to peak concentration, area under the curve from 0 to 72 hr and the elimination T1/2 for the terminal portion of the plasma concentration vs. time curve for TFPNO were found to be 3.7 +/- 0.7 ng/ml, 3.2 +/- 0.4 hr, 51.1 +/- 9.8 ng X hr/ml and 26.9 +/- 14.0 hr, respectively, whereas the corresponding values for TFP were found to be 2.7 +/- 0.7, 3.1 +/- 0.8, 42.2 +/- 6.9 and 21.9 +/- 6.5, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Radioimmunoassay for the 7-hydroxy metabolite of trifluoperazine and its application to a kinetic study in human volunteers.

A hapten derivative of the 7-hydroxy metabolite of trifluoperazine, 7-hydroxy-10-[[3-[4-(2-carboxyethyl)-1-piperazinyl]propyl]]-2 -tri-fluromethyl-10H-phenothiazine, was synthesized and coupled to bovine serum albumin. Immunization of New Zealand white rabbits with this drug-protein conjugate resulted in the production of antisera, one of which was subsequently utilized in the development of an RIA procedure. The described RIA for the first time enables the quantitation of the 7-hydroxy metabolite of trifluoperazine in human plasma after oral administration of single and therapeutic doses of trifluoperazine, in which 20 pg of the nonconjugated 7-hydroxy metabolite in 200 microL of plasma can be measured with a CV of less than 3% in B/Bo readings. Similar results were obtained by this assay procedure with or without the selective extraction of the 7-hydroxy metabolite and in the presence or absence of a large excess of trifluoperazine and other suspected major metabolites, such as the sulfoxide and N4'-oxide metabolites. This RIA procedure, together with a previously developed RIA for trifluoperazine, was used to directly determine plasma concentrations of trifluoperazine and its 7-hydroxy metabolite after administration of a single 5-mg oral dose of trifluoperazine to six healthy male volunteers. The mean +/- SD for the peak concentration (Cmax), the time to Cmax, the area under the curve from 0 to 24 h and, the apparent terminal elimination half-life for the 7-hydroxy metabolite were found to be 0.86 +/- 0.2 ng/mL, 6.2 +/- 1.6 h, 11.1 +/- 4.9 ng . h/mL, and 10.6 +/- 5.7 h, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of trifluoperazine (Stellazine) on Hymenolepis diminuta in vitro.

The influence of the phenothiazine trifluoperazine (Stellazine) on the rat tapeworm Hymenolepis diminuta was examined. The parasite was incubated in glucose-containing Krebs-Ringer media (pH 7.4) at 37 degrees C which included Ca2+ or EGTA and a range of trifluoperazine concentrations (0-2 mM). Release of soluble protein and lactate dehydrogenase activity were taken as measures of release of cytosolic components. The release of lactate dehydrogenase depended on drug concentration, maximum levels occurring at 2 mM trifluoperazine, this corresponded to 2% of the total lactate dehydrogenase present in the cestode. The effect of phenothiazines of differing lipophilicity were compared, and for trifluoperazine sulfoxide only minimal amounts of lactate dehydrogenase activity and protein were released. These values were similar to those obtained when H. diminuta was incubated in drug-free media. Our findings suggest that the integrity of the parasite is related to its calmodulin content. The potential cestocidal properties of trifluoperazine are considered.

Role of calmodulin in blastocyst formation in the mouse.

Preimplantation mouse embryos were recovered by flushing the oviducts on Day 3 at 09:30-10:00 h, 15:30-16:00 h and 21:30-22:00 h: When placed in culture for 48 h, 79% of the 4-8 cell embryos recovered at 09:30-10:00 h developed into blastocysts, but a large number of these embryos failed to form blastocysts when exposed to trifluoperazine, a calmodulin antagonist, at 0.5 or 0.6 microM in culture. About 45% of the embryos recovered at 15:30-16:00 h were compacted and blastocyst formation was again markedly depressed in the presence of the drug. Advanced compacted embryos recovered at 21:30-22:00 h showed normal development into blastocysts in the presence of 0.6 microM-trifluoperazine. Trifluoperazine sulphoxide (the inactive form of trifluoperazine) at 0.6 or 1.2 microM concentration had no effect on blastocyst formation of uncompacted embryos recovered at 09:30-10:00 h. These embryos and those recovered at 21:30-22:00 h and developed into blastocysts in the presence of trifluoperazine were transferred to Day-4 pseudopregnant mice and healthy young were born. When exposed to calcium-free medium or medium containing trifluoperazine all compacted embryos recovered at 18:30 h became decompacted; development to the blastocyst stage was normal in medium alone but reduced when trifluoperazine was added. Compacted embryos recovered at 23:00 h showed 100% decompaction in the calcium-free medium but completely failed to decompact in the presence of 0.6 microM-trifluoperazine. We suggest that extracellular calcium is essential for the continuance of compaction, while intracellular calcium is required only during the initial phase of this process.

Effects of phenothiazines on inhibition of plasma membrane ATPase and hyperpolarization of cell membranes in the yeast Saccharomyces cerevisiae.

The transmembranal potential, in Saccharomyces cerevisiae, has been calculated from the distribution ratio of the lipophilic cation tetraphenylphosphonium (TPP+) between the intracellular and extracellular water. Trifluoperazine at concentrations of 10 to 50 microM, caused a substantial increase in the membrane potential (negative inside). This increase was observed only in the presence of a metabolic substrate and was eliminated by the addition of the protonophores 2,4-dinitrophenol and sodium azide, removal of glucose, replacement of glucose by the nonmetabolizable analog 3-O-methyl glucose, or by the addition of 100 mM KCl. An increase in 45CaCl2 accumulation from solutions of low concentrations (1 microM) was observed under all conditions where membrane potential was increased. Proton ejection activity was monitored by measurements of the rates of the decrease in the pH of unbuffered cell suspensions in the presence of glucose. Trifluoperazine inhibited the changes in medium pH; this inhibition was not the result of an increase in the permeability of cell membranes to protons since in the absence of glucose, trifluoperazine did not cause a change in the rate of pH change generated by proton influx. The activity of plasma membrane ATPase was measured in crude membrane preparations in the presence of sodium azide to inhibit mitochondrial ATPase. Trifluoperazine strongly inhibited the activity of the plasma membrane ATPase. The effect of phenothiazines on transport and on membrane potential reported in this study and in the previous one (Eilam, Y. (1983) Biochim. Biophys. Acta 733, 242-248) were observed only in the presence of a metabolic substrate. The possibility that energy is required for the uptake of phenothiazines into the cells was eliminated by results showing energy-independent uptake of [3H]chlorpromazine. The results strongly suggest that phenothiazines activate energy-dependent K+-extrusion pumps, which lead to increased membrane potential. Increased influx of calcium seems to be energized by membrane potential, and therefore stimulated under all conditions where membrane potential is increased. The analog which does not bind to calmodulin, trifluoperazine sulfoxide, had no effect on the cells, but the involvement of calmodulin in the processes altered by trifluoperazine cannot as yet, be determined.

Effect of formulation of intramuscular injections of phenothiazines on duration of activity.

Trifluoperazine and pericyazine were formulated using both the hydrochloride and embonate salts, and some comparisons were made with the activity of fluphenazine salt and ester formulations. Simple solutions in polyethylene glycol, gelled aqueous solutions, nonaqueous suspensions, multiple emulsions, and microencapsulated preparations were formulated, and their duration of activity was tested in dogs. While the multiple emulsion system showed some promise, a nylon microcapsule system produced significant prolonged activity of the drug after deep intramuscular injection.