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Expert Opinion on Therapeutic Targets Jun 2017Tuberculosis (TB) is highly dangerous due to the development of resistance to first-line drugs. Moreover, Mycobacterium tuberculosis (Mtb) has also developed resistance... (Review)
Review
Tuberculosis (TB) is highly dangerous due to the development of resistance to first-line drugs. Moreover, Mycobacterium tuberculosis (Mtb) has also developed resistance to newly approved antitubercular drug bedaquiline. This necessitates the search for drugs acting on newer molecular targets. The energy metabolism of mycobacteria is the prime focus for the discovery of novel antitubercular drugs. Targeting type-2 NADH dehydrogenase (NDH-2) involved in the production of respiratory ATP could, therefore, be effective in treating the disease. Areas covered: This review describes the energetics of mycobacteria and the role of NDH-2 in ATP synthesis. Special attention has been given for genetic and chemical validations of NDH-2 as a molecular target. The reported kinetics and crystal structures of NDH-2 have been given in detail for better understanding of the enzyme. Expert opinion: NDH-2 is an essential enzyme for ATP synthesis and has a potential role in dormancy and persistence of Mtb. The human counterpart lacks this enzyme and hence NDH-2 inhibitors could have more clinical importance. Phenothiazines are potent inhibitor of NDH-2 and are effective against both drug-susceptible and drug-resistant Mtb. Thus, it is highly desirable to optimize phenothiazine class of compounds for the development of next generation anti-TB drugs.
Topics: Animals; Anti-Bacterial Agents; Antitubercular Agents; Drug Design; Drug Discovery; Drug Resistance, Neoplasm; Humans; Molecular Targeted Therapy; Mycobacterium tuberculosis; NADH Dehydrogenase; Phenothiazines; Tuberculosis
PubMed: 28472892
DOI: 10.1080/14728222.2017.1327577 -
International Journal of Biological... Sep 2019Life manifestation is mainly based on biopolymer-ligand molecular recognition; therefore, the elucidation of energy and speed associated with protein-ligand binding is...
Life manifestation is mainly based on biopolymer-ligand molecular recognition; therefore, the elucidation of energy and speed associated with protein-ligand binding is strategic in understanding and modulating biological systems. In this study, the interactions between methylene blue (MB) or azure A (AZA) dyes and bovine lactoferrin (BLF) were investigated by surface plasmon resonance, fluorescence spectroscopy, and isothermal titration microcalorimetry. Despite the molecular similarities between the dyes, the BLF-AZA binding thermodynamic parameters (ΔG = -30.50 and ΔH = 10.8 (kJ·mol)) were higher in magnitude than those of the BLF-MB systems (ΔG = -27.3 and ΔH = 5.72 (kJ·mol)). To increase the systems' entropy (TΔS = 41.3 and TΔS = 33.0 (kJ·mol)), the hydrophobic interactions must outweigh the electrostatic repulsion, thereby promoting BLF-dye binding. The activation complex formation (E = 33, E = 32, ∆H = 31, ∆H = 30, ∆G = 51.84, ∆G = 50.7, T∆S = -21, T∆S = -21 (kJ·mol)), owing to free BLF and MB (or AZA) associations, was not affected by the dye chemical structure, while for the thermodynamically stable BLF-dye complex dissociation, the same energetic parameters (E = 16, E = 6.4, ∆H = 14, ∆H = 3.9, ∆G = 81.4, ∆G = 74.93, T∆S = -68, T∆S = -71.0 (kJ·mol)) were considerably affected by the number of methyl groups. Our results may be very useful to determine binding processes controlled by kinetic parameters, as well as to optimize the application of these photosensitive dyes in biological systems.
Topics: Azure Stains; Coloring Agents; Hydrogen-Ion Concentration; Kinetics; Lactoferrin; Methylene Blue; Protein Binding; Surface Plasmon Resonance; Thermodynamics
PubMed: 31207326
DOI: 10.1016/j.ijbiomac.2019.06.097 -
Identification of phenothiazine derivatives as UHM-binding inhibitors of early spliceosome assembly.Nature Communications Nov 2020Interactions between U2AF homology motifs (UHMs) and U2AF ligand motifs (ULMs) play a crucial role in early spliceosome assembly in eukaryotic gene regulation. UHM-ULM...
Interactions between U2AF homology motifs (UHMs) and U2AF ligand motifs (ULMs) play a crucial role in early spliceosome assembly in eukaryotic gene regulation. UHM-ULM interactions mediate heterodimerization of the constitutive splicing factors U2AF65 and U2AF35 and between other splicing factors that regulate spliceosome assembly at the 3' splice site, where UHM domains of alternative splicing factors, such as SPF45 and PUF60, contribute to alternative splicing regulation. Here, we performed high-throughput screening using fluorescence polarization assays with hit validation by NMR and identified phenothiazines as general inhibitors of UHM-ULM interactions. NMR studies show that these compounds occupy the tryptophan binding pocket of UHM domains. Co-crystal structures of the inhibitors with the PUF60 UHM domain and medicinal chemistry provide structure-activity-relationships and reveal functional groups important for binding. These inhibitors inhibit early spliceosome assembly on pre-mRNA substrates in vitro. Our data show that spliceosome assembly can be inhibited by targeting UHM-ULM interactions by small molecules, thus extending the toolkit of splicing modulators for structural and biochemical studies of the spliceosome and splicing regulation.
Topics: Alternative Splicing; Humans; Phenothiazines; Protein Binding; Protein Domains; RNA Precursors; RNA Splicing Factors; Repressor Proteins; Spliceosomes; Splicing Factor U2AF
PubMed: 33159082
DOI: 10.1038/s41467-020-19514-1 -
Journal of Applied Microbiology Nov 2021As options to treat recalcitrant bacterial infections which are increasingly limited due to multidrug-resistant strains, searching for new, effective antibacterial...
AIM
As options to treat recalcitrant bacterial infections which are increasingly limited due to multidrug-resistant strains, searching for new, effective antibacterial compounds is necessary. One strategy is to generate treatment alternatives by drug repurposing.
METHODS AND RESULTS
In this work, phenotypic microarrays were used for the screening of miscellaneous compounds against the growth and biofilm formation of Acinetobacter baumannii, an important emergent multidrug-resistant opportunistic pathogen. The results showed that the phenothiazine derivatives, such as promethazine, trifluoperazine, thioridazine, and chlorpromazine, inhibited the growth of antibiotic-sensitive and multidrug-resistant strains (showing minimal inhibitory concentrations ranging from 0·05 to 0·6 g l and minimal bactericidal concentrations ranging from 0·1 to 2·5 g l ). All phenothiazine derivatives were active against biofilm cells (with minimal biofilm eradication concentrations ranging from 0·5 to >3 g l ). Chlorpromazine promoted reactive oxigen species (ROS) production, and cell membrane and DNA damage. Chlorpromazine showed synergy with antibiotics such as ceftazidime, meropenem, and colistin and was an effective treatment for experimentally infected Galleria mellonella when combined with ceftazidime.
CONCLUSIONS
It was demonstrated that phenothiazine derivatives, especially chlorpromazine, are drugs with attractive antibacterial properties against nosocomial MDR strains of A. baumannii, by generating ROS and cell membrane and DNA damage.
SIGNIFICANCE AND IMPACT OF THE STUDY
The present study indicates that repurposing phenothiazine derivatives for treating recalcitrant infections by A. baumannii could be promising.
Topics: Acinetobacter Infections; Acinetobacter baumannii; Anti-Bacterial Agents; Drug Resistance, Multiple, Bacterial; Drug Synergism; Humans; Microbial Sensitivity Tests; Phenothiazines
PubMed: 33884726
DOI: 10.1111/jam.15109 -
Scientific Reports Jun 2023Assessing the in vitro toxicity of compounds on cell cultures is an important step during the screening of candidate molecules for diverse applications. Among the...
Assessing the in vitro toxicity of compounds on cell cultures is an important step during the screening of candidate molecules for diverse applications. Among the strategies employed to determine cytotoxicity, MTT, neutral red, and resazurin are commonly used. Methylene blue (MB), a phenothiazinium salt, has several uses, such as dye, redox indicator, and even as treatment for human disease and health conditions, such as malaria and methemoglobinemia. However, MB has only been sparsely used as a cellular toxicity indicator. As a viability indicator, MB is mostly applied to fixed cultures at high concentrations, especially when compared to MTT or neutral red. Here we show that MB and its related compounds new methylene blue (NMB), toluidine blue O (TBO), and dimethylmethylene blue (DMMB) can be used as cytotoxicity indicators in live (non-fixed) cells treated for 72 h with DMSO and cisplatin. We compared dye uptake between phenothiazinium dyes and neutral red by analyzing supernatant and cell content via visible spectra scanning and microscopy. All dyes showed a similar ability to assess cell toxicity compared to either MTT or neutral red. Our method represents a cost-effective alternative to in vitro cytotoxicity assays using cisplatin or DMSO, indicating the potential of phenothiazinium dyes for the screening of candidate drugs and other applications.
Topics: Humans; Coloring Agents; Phenothiazines; Cisplatin; Neutral Red; Dimethyl Sulfoxide; Methylene Blue
PubMed: 37353536
DOI: 10.1038/s41598-023-36721-0 -
Antiviral Research Dec 2023Coronavirus (CoV) replication requires efficient cleavage of viral polyproteins into an array of non-structural proteins involved in viral replication, organelle...
Coronavirus (CoV) replication requires efficient cleavage of viral polyproteins into an array of non-structural proteins involved in viral replication, organelle formation, viral RNA synthesis, and host shutoff. Human CoVs (HCoVs) encode two viral cysteine proteases, main protease (M) and papain-like protease (PL), that mediate polyprotein cleavage. Using a structure-guided approach, a phenothiazine urea derivative that inhibits both SARS-CoV-2 M and PL protease activity was identified. In silico docking studies also predicted the binding of the phenothiazine urea to the active sites of structurally similar M and PL proteases from distantly related alphacoronavirus, HCoV-229 E (229 E), and the betacoronavirus, HCoV-OC43 (OC43). The lead phenothiazine urea derivative displayed broad antiviral activity against all three HCoVs tested in cellulo. It was further demonstrated that the compound inhibited 229 E and OC43 at an early stage of viral replication, with diminished formation of viral replication organelles, and the RNAs that are made within them, as expected following viral protease inhibition. These observations suggest that the phenothiazine urea derivative readily inhibits viral replication and may broadly inhibit proteases of diverse coronaviruses.
Topics: Humans; Peptide Hydrolases; SARS-CoV-2; Papain; Viral Proteases; Phenothiazines; Protease Inhibitors; Antiviral Agents
PubMed: 38008194
DOI: 10.1016/j.antiviral.2023.105758 -
Journal of Biomolecular NMR Jul 2021We used NMR to show that the antipsychotic phenothiazine drugs promazine and promethazine bind to GDP-KRAS. Promazine also binds to oncogenic GDP-KRAS(G12D), and to wild...
We used NMR to show that the antipsychotic phenothiazine drugs promazine and promethazine bind to GDP-KRAS. Promazine also binds to oncogenic GDP-KRAS(G12D), and to wild type GppNHp-KRAS. A panel of additional phenothiazines bind to GDP-KRAS but with lower affinity than promazine or promethazine. Binding is most dependent on substitutions at C-2 of the tricyclic phenothiazine ring. Promazine was used to generate an NMR-driven HADDOCK model of the drug/GDP-KRAS complex. The structural model shows the tricyclic phenothiazine ring of promazine associates with the hydrophobic pocket p1 that is bordered by the central β sheet and Switch II in KRAS. Binding appears to stabilize helix 2 in a conformation that is similar to that seen in KRAS bound to other small molecules. Association of phenothiazines with KRAS may affect normal KRAS signaling that could contribute to multiple biological activities of these antipsychotic drugs. Moreover, the phenothiazine ring represents a new core scaffold on which to design modulators of KRAS activity.
Topics: Amino Acid Substitution; Antipsychotic Agents; Humans; Models, Molecular; Mutation, Missense; Nuclear Magnetic Resonance, Biomolecular; Phenothiazines; Protein Binding; Protein Conformation, beta-Strand; Proto-Oncogene Proteins p21(ras)
PubMed: 34176062
DOI: 10.1007/s10858-021-00371-z -
Brain Research Jul 2021After ischemic stroke, the increased catabolism of glucose (hyperglycolysis) results in the production of reactive oxygen species (ROS) via nicotinamide adenine...
BACKGROUND
After ischemic stroke, the increased catabolism of glucose (hyperglycolysis) results in the production of reactive oxygen species (ROS) via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). A depressive or hibernation-like effect of C + P on brain activity was reported to induce neuroprotection. The current study assesses the effect of C + P on hyperglycolysis and NOX activation.
METHODS
Adult male Sprague-Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) followed by 6 or 24 h of reperfusion. At the onset of reperfusion, rats received C + P with or without temperature control, or phloretin [glucose transporter (GLUT)-1 inhibitor], or cytochalasin B (GLUT-3 inhibitor). We detected brain ROS, apoptotic cell death, and ATP levels along with HIF-1α expression. Cerebral hyperglycolysis was measured by glucose, protein expression of GLUT-1/3, and phosphofructokinase-1 (PFK-1), as well as lactate and lactate dehydrogenase (LDH) at 6 and 24 h of reperfusion. The enzymatic activity of NOX and protein expression of its subunits (gp91) were detected. Neural SHSY5Y cells were placed under 2 h of oxygen-glucose deprivation (OGD) followed by reoxygenation for 6 and 24 h with C + P treatment. Cell viability and protein levels of HIF-1α, GLUT-1/3, PFK-1, LDH, and gp91 were measured. A HIF-1α overexpression vector was transfected into the cells, and then protein levels of HIF-1α, GLUT-1/3, PFK-1, and LDH were quantitated. In sham-operated rats and control cells, the protein levels of HIF-1α, GLUT-1/3, PFK-1, LDH, and gp91 were measured at 6 and 24 h after C + P administration.
RESULTS
C + P reduced the protein elevations after stroke in HIF-1α, glycolytic enzymes, as well as in ROS, cell death, glucose and lactate, but raised ATP levels in the brain. In ischemic rats exposed to GLUT-1/3 inhibitors, ROS, cell death, glucose, and lactate were all decreased, as well as GLUT-1, GLUT-3, LDH, and PFK-1 protein levels. C + P decreased ischemia-induced NOX activation by reducing the enzymatic activity and protein expression of the NOX subunit gp91, as was observed in the presence of GLUT-1/3 inhibitors. These markers were significantly decreased following C + P administration with the induced hypothermia, while C + P administration with temperature control at 37 °C induced lesser protection after ischemia stroke. In the OGD/reoxygenation model, C + P treatment increased cell viability and diminished protein levels of HIF-1α, GLUT-1, GLUT-3, PFK-1, LDH, and gp91. However, in OGD with HIF-1α overexpression, C + P was unable to effectively reduce the upregulated GLUT-1, GLUT-3, and LDH. In normal conditions, C + P reduced HIF-1α and the levels of key glycolytic enzymes depending on its pharmacological effect.
CONCLUSION
C + P, partially depending on hypothermia, attenuates hyperglycolysis and NOX activation through HIF-1α regulation.
Topics: Animals; Chlorpromazine; Glucose; Glucose Transporter Type 1; Glucose Transporter Type 3; Glycolysis; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Infarction, Middle Cerebral Artery; Ischemic Stroke; L-Lactate Dehydrogenase; Male; NADPH Oxidase 2; Phosphofructokinase-1; Promethazine; Rats; Rats, Sprague-Dawley
PubMed: 33811844
DOI: 10.1016/j.brainres.2021.147463 -
Archives of Toxicology Oct 2017Phenyl valerate is used for detecting and measuring neuropathy target esterase (NTE) and has been used for discriminating esterases as potential target in hen model of...
Phenyl valerate is used for detecting and measuring neuropathy target esterase (NTE) and has been used for discriminating esterases as potential target in hen model of organophosphorus delayed neuropathy. In previous studies we observed that phenyl valerate esterase (PVase) activity of an enzymatic fraction in chicken brain might be due to a butyrylcholinesterase protein (BuChE), and it was suggested that this enzymatic fraction could be related to the potentiation/promotion phenomenon of the organophosphate-induced delayed neuropathy (OPIDN). In this work, PVase activity of purified human butyrylcholinesterase (hBuChE) is demonstrated and confirms the novel observation that a relationship of BuChE with PVase activities is also relevant for humans, as is, therefore the potential role in toxicity for humans. The K and catalytic constant (k) were estimated as 0.52/0.72 µM and 45,900/49,200 min respectively. Furthermore, this work studies the inhibition by preincubation of PVase and cholinesterase activities of hBuChE with irreversible inhibitors (mipafox, iso-OMPA or PMSF), showing that these inhibitors interact similarly in both activities with similar second-order inhibition constants. Acethylthiocholine and phenyl valerate partly inhibit PVase and cholinesterase activities, respectively. All these observations suggest that both activities occur in the same active center. The interaction with a reversible inhibitor (ethopropazine) showed that the cholinesterase activity was more sensitive than the PVase activity, showing that the sensitivity for this reversible inhibitor is affected by the nature of the substrate. The present work definitively establishes the capacity of BuChE to hydrolyze the carboxylester phenyl valerate using a purified enzyme (hBuChE). Therefore, BuChE should be considered in the research of organophosphorus targets of toxicity related with PVase proteins.
Topics: Acetylcholine; Butyrylcholinesterase; Carboxylic Ester Hydrolases; Cholinesterase Inhibitors; Humans; Hydrolysis; Isoflurophate; Phenothiazines; Phenylmethylsulfonyl Fluoride; Tetraisopropylpyrophosphamide; Valerates
PubMed: 28299395
DOI: 10.1007/s00204-017-1946-5 -
Journal of Advanced Research Mar 2022Cancer is a big challenge of the 21 century, whose defeat requires efficient antitumor drugs.
INTRODUCTION
Cancer is a big challenge of the 21 century, whose defeat requires efficient antitumor drugs.
OBJECTIVES
The paper aims to investigate the synergistic effect of two structural building blocks, phenothiazine and poly(ethylene glycol), towards efficient antitumor drugs.
METHODS
Two PEGylated phenothiazine derivatives were synthetized by attaching poly(ethylene glycol) of 550 Da to the nitrogen atom of phenothiazine by ether or ester linkage. Their antitumor activity has been investigated on five human tumour lines and a mouse tumor line as well, by determination of IC50. The toxicity was determined by measuring the LD50 in BALB/c mice by the sequential method and the antitumor potential was measured by the tumours growth test. The antitumor mechanism was investigated by complexation studies of zinc and magnesium ions characteristic to the farnesyltransferase enzyme, by studies of self-aggregation in the cells proximity and by investigation of the antitumor properties of the acid species resulted by enzymatic cleavage of the PEGylated derivatives.
RESULTS
The two compounds showed antitumor activity, with IC50 against mouse colon carcinoma cell line comparable with that of the traditional antitumor drugs 5-Fluorouracil and doxorubicin. The phenothiazine PEGylation resulted in a significant toxicity diminishing, the LD50 in BALB/c mice increasing from 952.38 up to 1450 mg/kg, in phenothiazine equivalents. Both compounds inflicted a 92% inhibition of the tumour growth for doses much smaller than LD50. The investigation of the possible tumour inhibition mechanism suggested the nanoaggregate formation and the cleavage of ester bonds as key factors for the inhibition of cancer cell proliferation and biocompatibility improvement.
CONCLUSION
Phenothiazine and PEG building blocks have a synergetic effect working for both tumour growth inhibition and biocompatibility improvement. All these findings recommend the PEGylated phenothiazine derivatives as a valuable workbench for a next generation of antitumor drugs.
Topics: Animals; Antineoplastic Agents; Antipsychotic Agents; Esters; Farnesyltranstransferase; Mice; Phenothiazines; Polyethylene Glycols
PubMed: 35499049
DOI: 10.1016/j.jare.2021.07.003