High content phenotypic screening identifies serotonin receptor modulators with selective activity upon breast cancer cell cycle and cytokine signaling pathways.

Heterogeneity in disease mechanisms between genetically distinct patients contributes to high attrition rates in late stage clinical drug development. New personalized medicine strategies aim to identify predictive biomarkers which stratify patients most likely to respond to a particular therapy. However, for complex multifactorial diseases not characterized by a single genetic driver, empirical approaches to identifying predictive biomarkers and the most promising therapies for personalized medicine are required. In vitro pharmacogenomics seeks to correlate in vitro drug sensitivity testing across panels of genetically distinct cell models with genomic, gene expression or proteomic data to identify predictive biomarkers of drug response. However, the vast majority of in vitro pharmacogenomic studies performed to date are limited to dose-response screening upon a single viability assay endpoint. In this article we describe the application of multiparametric high content phenotypic screening and the theta comparative cell scoring method to quantify and rank compound hits, screened at a single concentration, which induce a broad variety of divergent phenotypic responses between distinct breast cancer cell lines. High content screening followed by transcriptomic pathway analysis identified serotonin receptor modulators which display selective activity upon breast cancer cell cycle and cytokine signaling pathways correlating with inhibition of cell growth and survival. These methods describe a new evidence-led approach to rapidly identify compounds which display distinct response between different cell types. The results presented also warrant further investigation of the selective activity of serotonin receptor modulators upon breast cancer cell growth and survival as a potential drug repurposing opportunity.

Modification of the thermal unfolding pathways of myoglobin upon drug interaction in different aqueous media.

In this work, we have analyzed the influence of two structurally related phenothiazine drugs, promazine and triflupromazine hydrochlorides, when bound to myoglobin, a model protein, and how the drug concentration and solution conditions may affect the denaturation process of this protein. In this manner, we derive the thermodynamic quantities of the unfolding process by using a spectroscopic technique such as UV-vis spectroscopy at different drugs concentrations and at pH 2.5, 5.5, and 9.0. To do this, a thermodynamic model was used which included experimental data corresponding to the pre- and post-transition into the observable transition. It has been found that both drugs play a destabilizing role for the protein, at least at low concentrations. In addition, at acidic pH and higher drug concentrations, a stabilizing effect can be observed, which may be related to the formation of some type of protein refolding, subsequent aggregation, or both. The reason for this behavior has been suggested to be the different protein conformations at acidic pH, the increase of solvent-exposed hydrophobic and hydrophilic residues after denaturation and/or binding, and the different strength of drug-protein interactions when changing the solution conditions. For this reason, thermodynamic quantities such as Gibbs energies, DeltaG, and entropies of unfolding, DeltaS(m), increase as the solution pH increases provided that additional solvent-exposed hydrophobic residues are present, which were previously buried at room temperature. Moreover, the larger binding affinity at pH 9.0 due to enhanced electrostatic interactions between protein and drug molecules (drug and protein differ in their net electrical charge) additionally collaborates to this residue exposition to solvent as a consequence of the alteration of protein conformation as due to drug binding. Comparison of thermodynamic data between promazine and triflupromazine hydrochlorides also shows that drug-protein affinity and hydrophobicity also affect the thermodynamic denaturation parameters.

Effects of inorganic ions on the binding of triflupromazine and chlorpromazine to bovine serum albumin studied by spectrometric methods.

The effects of inorganic salts, NaCl, NaBr, NaI, Na2SO4, KCl, KBr, KI, on the binding constants (Ks) of psychotropic phenothiazine drugs, triflupromazine (TFZ) and chlorpromazine, to bovine serum albumin (BSA) were examined by using second-derivative spectrophotometry. All of the salts examined, with the exception of Na2SO4, decreased the K values significantly, depending on the concentration of the salt, e.g., the decrease in the K values of both drugs were about 40% for 0.1 M NaCl. The results obtained with Na2SO4 indicated that neither Na+ nor SO4(2-) had any affect on the binding of the phenothiazines to BSA. Based on the Na2SO4 results and the finding that the effect of each potassium salt on binding was quite similar to that of the corresponding sodium salt, the effects of these halogen salts can be considered to be derived from their anions, although the phenothiazines are positively charged at pH 7.4. The effectiveness of the anions was determined to occur in the following order: I->>Br->Cl-; these results coincided with the published order of the binding affinity of these anions to albumin. The 19F-NMR spectra of TFZ in the presence of each of these halogen salts revealed a concentration-dependent decrease in the intensity of the signal at 13.8 ppm that had previously been assigned to the TFZ bound to Site II. Consequently, the effects of these anions on the binding of positively charged phenothiazine drugs are thought to be local steric effects caused by the binding of these anions to Site II.

Effects of phosphatidylserine and phosphatidylethanolamine content on partitioning of triflupromazine and chlorpromazine between phosphatidylcholine-aminophospholipid bilayer vesicles and water studied by second-derivative spectrophotometry.

To assess the affinity of psychotropic phenothiazine drugs, triflupromazine (TFZ) and chlorpromazine (CPZ), for the membranes of central nervous system and the other organs in the body, the partition coefficients (Kps) of these drugs to phosphatidylcholine (PC)-phosphatidylserine (PS) and PC-phosphatidylethanolamine (PE) small and large unilamellar vesicles (SUV, LUV) were examined by a second-derivative spectrophotometric method, since PS is abundantly contained in the membranes of the central nervous system and PE is distributed widely in the membranes of the organs in the body. Size and preparation methods of the vesicles did not affect the Kp values at each aminophospholipid content suggesting that the partition of the phenothiazine drugs was not affected by the structural differences in the vesicles such as their curvature or asymmetric distribution of the phospholipids between the outer and inner layers of the bilayer membranes. However, the Kp values of both drugs increased remarkably according to the PS content in the bilayer membranes, i.e., the Kp values for the vesicles of 30 mol% PS content were about 3 times of that for the vesicles of PC alone, while both Kp values slightly reduced with the increase in the content of PE in the bilayer membranes of PC-PE vesicles. The results indicate that both drugs have higher affinity for the PC-PS bilayer membranes than for the PC and PC-PE membranes, which can offer an evidence for the fact that TFZ and CPZ are predominantly distributed and accumulated in the brain and nerve cell membranes that contain PS abundantly.

Dissociation constants of phenothiazine drugs incorporated in phosphatidylcholine bilayer of small unilamellar vesicles as determined by carbon-13 nuclear magnetic resonance spectrometric titration.

The dissociation constants (pKms) of the phenothiazine drugs promazine, chlorpromazine, and triflupromazine, incorporated in the phosphatidylcholine (PC) bilayer of small unilamellar vesicles (SUV), were investigated by a 13C nuclear magnetic resonance (NMR) titration method employing their N-13CH3 (ionizable group) labelled derivatives. Use of the labelled drugs enabled direct observations of the ionization equilibrium of the N-dimethyl group. A second derivative spectrophotometric study proved that 95-98% of the phenothiazine species in the sample solutions (200 microM phenothiazine in the presence of 27 mM PC SUV) were incorporated into the PC bilayer, which simplified the calculation of pKm values by allowing that the phenothiazines in the aqueous phase could be neglected. The pKm values were calculated from the chemical shift dependence of the N-dimethyl 13C NMR signal on the pH value of sample solutions. The pKm values obtained were smaller than those measured in aqueous solutions by about one unit. The existence of cholesterol (30 mol%) in the PC bilayer showed little effect on the pKm values, suggesting that cholesterol in the bilayer does not largely affect the interfacial region where the N-dimethyl group of the incorporated phenothiazines is located. The results offered clear evidence for the pKm decrease and provided their precise values.

Pharmacological profile of neuroleptics at human monoamine transporters.

Using radioligand binding techniques, we determined the equilibrium dissociation constants (K(D)) for 37 neuroleptics and one metabolite of a neuroleptic (haloperidol metabolite) for the human serotonin, norepinephrine, and dopamine transporters with [3H]imipramine, [3H]nisoxetine, and [3H]WIN35428, respectively. Among neuroleptics, the four most potent compounds at the human serotonin transporter were triflupromazine, fluperlapine, chlorpromazine, and ziprasidone (K(D) 24-39 nM); and at the norepinephrine transporter, chlorpromazine, zotepine, chlorprothixene, and promazine (K(D) 19-25 nM). At the human dopamine transporter, only pimozide (K(D) = 69+/-3) ziprasidone (K(D) = 76+/-5) had notable potency. These data may be useful in predicting therapeutic and adverse effects, including drug interactions of neuroleptics.

A kinetic study of the generation and decomposition of some phenothiazine free radicals formed during enzymatic oxidation of phenothiazines by peroxidase-hydrogen peroxide.

A kinetic study of the oxidation of four different phenothiazines (Pts) by peroxidase-hydrogen peroxide was carried out. The free radical formed during the enzymatic oxidation suffers a non-enzymatic breakdown and the overall system was analysed and characterized. The non-enzymatic breakdown of the cation radical does not occur through a disproportionation mechanism but through a more complex mechanism. The kinetic parameters of the overall system were determined for the different Pts. These experimental data may serve in the understanding of the pharmacological action of Pts.

[Localization of two neurotropic molecules in cultured glial cells by ion microscopy]. / Localisation de deux molécules neurotropes dans les cellules gliales en culture par microscopie ionique.

The intracellular localization of two families of neurotropic drugs: flunitrazepam and flurazepam (benzodiazepine), triflupromazine and trifluoperazine (phenothiazine) has been studied by ion microscopy. The molecules have been incubated with C6 glioblastoma cells from rat origin and with astroglial primary cultures. The images of the intracellular distributions of the two drugs are easily obtained by selecting the fluorine atom of the molecules. The images obtained show that flunitrazepam and flurazepam, two drugs of the benzodiazepine group are mainly located to the nuclei, whereas triflupromazine and trifluoperazine, two phenothiazines are exclusively located inside the cytoplasm.