Phenothiazine Drugs Research Articles

Simultaneous determination of phenothiazine drugs and their metabolites residues in animal derived foods by high performance liquid chromatography tandem mass spectrometry

Due to their potential entry into the human body via the food chain, phenothiazine drugs and their metabolites pose specific health hazards, making their detection in edible tissues of paramount importance. In this research endeavor, a highly sensitive multi-residue analytical method has been devised for the detection of phenothiazine drugs and their metabolites in animal-derived food products, utilizing high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The preparation of samples involved the use of 1% formic acid in acetonitrile as the extraction solvent following sample weighing, with multiple extraction steps diligently performed to ensure optimal outcomes. The sample, post-extraction, was directly introduced into the purification column, yielding significant time savings and rendering the process more environmentally sustainable. This procedural step effectively eliminated interfering substances from the sample matrix, including proteins, salts, and phospholipids. Analysis of the extracted samples was conducted through HPLC-MS/MS, employing an electrospray ionization (ESI) ion source. A comprehensive panel of 20 phenothiazine drugs and their metabolites was successfully resolved within a mere 10-min chromatographic run. A robust linear correlation (R2 ≥ 0.99) was attained within a judiciously chosen range of concentrations. Method validation was meticulously conducted across multiple animal species, including trout, white shrimp, pork, beef and chicken. The developed analytical method has been effectively deployed for the screening and quantification of phenothiazine drugs in real-world samples sourced from commercial livestock and aquatic products.

Biological Applications, In Vitro Cytotoxicity, Cellular Uptake, and Apoptotic Pathway Studies Induced by Ternary Cu (II) Complexes Involving Triflupromazine with Biorelevant Ligands.

The novel mixed-ligand complexes derived from the parent antidepressant phenothiazine drug triflupromazine (TFP) were synthesized along with the secondary ligands glycine and histidine. [Cu(TFP)(Gly)Cl]·2H2O (1) and [Cu(TFP)(His)Cl]·2H2O (2) were examined for their in vitro biological properties. Cyclic voltammetry was used to study the binding of both complexes to CT-DNA. The two complexes were examined for antiviral, antiparasite, and anti-inflammatory applications. An in vitro cytotoxicity study on two different cancer cell lines, MCF-7, HepG2, and a normal cell line, HSF, shows promising selective cytotoxicity for cancer cells. An investigation of the cell cycle and apoptosis rates was evaluated by flow cytometry with Annexin V-FITC/Propidium Iodide (PI) staining of the treated cells. Gene expression and western blotting were carried out to determine the expression levels of the pro-apoptotic markers and the anti-apoptotic marker Bcl2. The tested complexes decreased cell viability and triggered apoptosis in human tumor cell lines. Molecular docking was also used to simulate Bcl2 inhibition. Finally, complex (2) has potent antitumor effects on human tumor cells, especially against HepG2 cells, as seen in the cellular drug uptake assay. Consequently, complex (2) may prove useful against cancer, especially liver cancer. For further understanding, it needs to be explored in vivo.

Modulation of NLRP3 inflammasome-related-inflammation via RIPK1/RIPK3-DRP1 or HIF-1α signaling by phenothiazine in hypothermic and normothermic neuroprotection after acute ischemic stroke

BackgroundInflammation and subsequent mitochondrial dysfunction and cell death worsen outcomes after revascularization in ischemic stroke. Receptor-interacting protein kinase 1 (RIPK1) activated dynamin-related protein 1 (DRP1) in a NLRPyrin domain containing 3 (NLRP3) inflammasome-dependent fashion and Hypoxia-Inducible Factor (HIF)-1α play key roles in the process. This study determined how phenothiazine drugs (chlorpromazine and promethazine (C + P)) with the hypothermic and normothermic modality impacts the RIPK1/RIPK3-DRP1 and HIF-1α pathways in providing neuroprotection. MethodsA total of 150 adult male Sprague-Dawley rats were subjected to 2 h middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion. 8 mg/kg of C + P was administered at onset of reperfusion. Infarct volumes, mRNA and protein expressions of HIF-1α, RIPK1, RIPK3, DRP-1, NLRP3-inflammation and cytochrome c-apoptosis were assessed. Apoptotic cell death, infiltration of neutrophils and macrophages, and mitochondrial function were evaluated. Interaction between RIPK1/RIPK3 and HIF-1α/NLRP3 were determined. In SH-SY5Y cells subjected to oxygen/glucose deprivation (OGD), the normothermic effect of C + P on inflammation and apoptosis were examined. ResultsC + P significantly reduced infarct volumes, mitochondrial dysfunction (ATP and ROS concentration, citrate synthase and ATPase activity), inflammation and apoptosis with and without induced hypothermia. Overexpression of RIPK1, RIPK3, DRP-1, NLRP3-inflammasome and cytochrome c-apoptosis were all significantly reduced by C + P at 33 °C and the RIPK1 inhibitor (Nec1s), suggesting hypothermic effect of C + P via RIPK1/RIPK3-DRP1pathway. When body temperature was maintained at 37 °C, C + P and HIF-1α inhibitor (YC-1) reduced HIF-1α expression, leading to reduction in mitochondrial dysfunction, NLRP3 inflammasome and cytochrome c-apoptosis, as well as the interaction of HIF-1α and NLRP3. These were also evidenced in vitro, indicating a normothermic effect of C + P via HIF-1α. ConclusionHypothermic and normothermic neuroprotection of C + P involve different pathways. The normothermic effect was mediated by HIF-1α, while hypothermic effect was via RIPK1/RIPK3-DRP1 signaling. This provides a theoretical basis for future precise exploration of hypothermic and normothermic neuroprotection.

Pyroptosis of Breast Cancer Stem Cells and Immune Activation Enabled by a Multifunctional Prodrug Photosensitizer

AbstractBreast cancer stem cells (CSCs) are responsible for the occurrence, resistance, recurrence, invasion, and metastasis of tumors. However, even trace amounts of CSCs may lead to tumor resistance and recurrence, which fundamentally reduces the therapeutic efficiency of numerous anticancer drugs. Thus, the development of a therapeutic agent that can reduce the tumorigenicity of CSCs and overcome tumor resistance and recurrence is essential. Here a novel multifunctional prodrug T‐P is reported as a photosensitizer, which links phenothiazine drug with the synthesized aggregation‐induced emission photosensitizer T‐C via an ester bond. Importantly, this photosensitizer is found to be able to induce the pyroptosis of breast CSCs as well as to activate their death pathway protein phosphatase 2A to inhibit CSCs and systemic anti‐tumor effects. T‐P can rapidly target mitochondria and overlap with lysosomes after mitochondrial escape, and it can cause mitochondrial and lysosomal dysfunction. It releases reactive oxygen species through photoactivation, triggering pyroptosis‐mediated strong anti‐tumor immune response. On the 5th day of in vivo therapy of breast cancer, the primary tumor is eliminated and the growth of distant tumors is also inhibited. This research would provide an impetus as well as a new strategy for CSCs‐targeted cancer photoimmunotherapy and beyond.

Machine learning-based fluorescence sensor array: Accurate discrimination, quantitative assay of phenothiazine drugs via versatile DNA probes

Phenothiazine drugs are widely used to treat psychotic diseases in humans. The phenothiazine ligand enables unique interaction between the drugs and DNA double helix. We discovered that it was impossible to select aptamer to selectively bind with phenothiazine drug. In this study, an alternative fluorescence sensor array was first developed to accurately discriminate four typical phenothiazine drugs based on machine learning. As the eight sensor elements, versatile DNA probes were designed based on different oligonucleotides, two unique fluorescence dyes, and diverse buffer solutions. The fingerprint data can offer the fluorescence response patterns against each phenothiazine drug. Linear discriminant analysis (LDA) can discriminate single one or mixture samples of phenothiazine drugs with 100% accuracy. Besides, one neural network model showed a good linear relationship between predicted and actual concentrations to quantitatively assay specific phenothiazine drugs. The proposed fluorescence sensor array presents a new way to accurately discriminate phenothiazine drugs in pharmacy or biology field.

Host-guest systems for the SAMPL9 blinded prediction challenge: phenothiazine as a privileged scaffold for binding to cyclodextrins.

Model systems are widely used in biology and chemistry to gain insight into more complex systems. In the field of computational chemistry, researchers use host-guest systems, relatively simple exemplars of noncovalent binding, to train and test the computational methods used in drug discovery. Indeed, host-guest systems have been developed to support the community-wide blinded SAMPL prediction challenges for over a decade. While seeking new host-guest systems for the recent SAMPL9 binding prediction challenge, which is the focus of the present PCCP Themed Collection, we identified phenothiazine as a privileged scaffold for guests of β cyclodextrin (βCD) and its derivatives. Building on this observation, we used calorimetry and NMR spectroscopy to characterize the noncovalent association of native βCD and three methylated derivatives of βCD with five phenothiazine drugs. The strongest association observed, that of thioridazine and one of the methyl derivatives, exceeds the well-known high affinity of rimantidine with βCD. Intriguingly, however, methylation of βCD at the 3 position abolished detectible binding for all of the drugs studied. The dataset has a clear pattern of entropy-enthalpy compensation. The NMR data show that all of the drugs position at least one aromatic proton at the secondary face of the CD, and most also show evidence of deep penetration of the binding site. The results of this study were used in the SAMPL9 blinded binding affinity-prediction challenge, which are detailed in accompanying papers of the present Themed Collection. These data also open the phenothiazines and, potentially, chemically similar drugs, such as the tricyclic antidepressants, as relatively potent binders of βCD, setting the stage for future SAMPL challenge datasets and for possible applications as drug reversal agents.

Prehemolytic impact of phenothiazine drugs on the attachment of spectrin network in red blood cells.

Chlorpromazine, thioridazine, and trifluoperazine are phenothiazine drugs that cause colloid-osmotic hemolysis of human erythrocytes by unknown mechanism. To clarify this mechanism, the impact of these drugs on the βsp (1.4 MHz) and γ1sp (9 MHz) dielectric relaxations was investigated. Each relaxation was shown to reduce its strength on the severing specific bridge that connects the spectrin network with the lipid membrane. For βsp relaxation, this is the spectrin-actin-glycophorin C bridge while for γ1sp relaxation this is the spectrin-ankyrin-band 3 bridge.

Ultrasensitive determination of 10 phenothiazine derivatives and their enantiomers in biological fluids by capillary electrophoresis with contactless conductivity detection

In this study, a simple, rapid, and ultrasensitive technique was developed to identify five pairs of phenothiazine drugs by using ultrasound-enhanced and surfactant-assisted dispersive liquid–liquid microextraction (UESA-DLLME), field-amplified sample injection with capillary electrophoresis (FASI-CE), and capacitively coupled capacitively coupled contactless conductivity detection (C4D). During the CE separation process, UESA-DLLME was used for sample clean-up and offline concentration, and FASI-CE was used for the online concentration of phenothiazine enantiomers. At baseline, the five pairs of phenothiazine enantiomer drugs required 18 min for separation. UESA-DLLME was then used to extract 0.01 mM Tween 80 at pH 10 from a sample solution (extraction solvent, 100 mL of dichloromethane). Subsequently, FASI was used to stack the sample solution (buffer, 30 mM 2-(N-morpholino)ethanesulfonic acid/aspartic acid, additive 4 mM hydroxypropyl-γ-cyclodextrin, pH 2.5), and C4D was used for signal detection (amplitude, 2 Vpp; frequency, 400 kHz). The results indicated that the linear range for quantifying all analyte enantiomers was 1.0–150 nM, with a coefficient of determination exceeding 0.99. In addition, the relative standard deviations in the migration time and peak areas for the 10 analytes were less than 3.2% and 7.2%, respectively. The proposed system has a limit of detection (LOD) for the 10 analytes at a signal-to-noise ratio of 3, ranging from 0.24 to 0.28 nM. The sensitivity enhancement, which compares the LOD0 (limit of detection in the normal method) to LOD1 (limit of detection achieved using the proposed UESA-DLLME-FASI-CE-C4D method), varies between approximately 1200 and 2000 for the 10 analytes. Analysis of the 10 separated analytes spiked in urine and serum samples revealed recovery rates of 88%–106% and 89%–105%, respectively. Therefore, this highly sensitive advanced technique was successfully used to analyze phenothiazine enantiomers in urine and serum samples.

Micellization of phenothiazine drug and nonionic surfactant TX-165 mixture in different composition and media: surface tension and UV-visible study

ABSTRACT Micelles serve as a replica for the investigation of bio-membranes as physicochemical association of drugs with micelles is envisioned as their interconnection with bio-membranes. Micellization properties of promethazine hydrochloride (PMT) with TX-165 (a nonionic surfactant) in aqueous, 50 mmolkg−1 NaCl and 500 mmolkg−1 urea solutions were analysed in detail using surface tension measuring tool. PMT is employed as an analgesic, anticholinergic, sedative, antihistaminic as well as antipsychotic drug. UV-visible spectroscopy is also utilised to evaluate PMT-TX-165 interactions in an aqueous solution. Pure constituent and their mixtures (PMT + TX-165) of each composition showed lower cmc value in NaCl than in the aqueous system, while urea-containing solutions showed higher cmc value. Increasing the mole fraction (α 1) of TX-165 in mixed systems increases the interaction between the employed constituents (PMT and TX-165). The evaluated Gibbs free energy values of singular, and mixed system in each media were attained negative that uncovering that each system depicted the spontaneity of micellization. Various thermodynamic parameters were examined and discussed thoroughly. A UV-visible measurement also confirms that the components interact in treaty with surface tension measurements. Based on the results of this study, TX-165 was designed in a forthright manner, which could be an efficient drug delivery vehicle for phenothiazine drugs.

Analysis of Some Phenothiazine Drugs and Their S-Oxides using Enzymatic Method of Cholinesterase Inhibition

In this work, a highly sensitive spectrophotometric method was developed for the determination of cholinesterase (AChE) inhibitors - phenothiazine derivatives (PhTs). The assay involves spectrophotometric measurement of a mixture of buffer, solutions of acetylcholine (Ach), a sample containing AChE, H2O2, and PhTs. The rate of the enzymatic hydrolysis of Ach reaction was determined by the tangent method of the linear part of the kinetic curve in the A (358 nm) – t-coordinate. Results from the determination of PhTs and S-oxide PhTs with the AChE regulated spectrophotometric system showed the limit of quantitation of 5 ng/mL (IE20) and a linear dynamic range from 5 to 30 ng/mL for Chlorpromazine, Promethazine and from 0.5 to 10 ng/mL for Chlorpromazine S-oxide, from 1 to 10 ng/mL for Promethazine S-oxide, 12 to 40 ng/mL for Thioridazine 2,5-disulfoxide respectively. RSD for concentrations of PhTs as low as 1.5·10-8 mol/L did not exceed 6.7 %, while their corresponding sulfoxides at 1.50·10-9 didn’t exceed +6.5 %. δ < +3.8 %. Findings in this work demonstrate that this method may be used for the determination of phenothiazine based drugs, and sensitive tests for rapid PhT monitoring without the addition of other exogenous catalysts

Pleiotropic actions of phenothiazine drugs are detrimental to Gram-negative bacterial persister cells

Bacterial persister cells are temporarily tolerant to bactericidal antibiotics but are not necessarily dormant and may exhibit physiological activities leading to cell damage. Based on the link between fluoroquinolone-mediated SOS responses and persister cell recovery, we screened chemicals that target fluoroquinolone persisters. Metabolic inhibitors (e.g., phenothiazines) combined with ofloxacin (OFX) perturbed persister levels in metabolically active cell populations. When metabolically stimulated, intrinsically tolerant stationary phase cells also became OFX-sensitive in the presence of phenothiazines. The effects of phenothiazines on cell metabolism and physiology are highly pleiotropic: at sublethal concentrations, phenothiazines reduce cellular metabolic, transcriptional, and translational activities; impair cell repair and recovery mechanisms; transiently perturb membrane integrity; and disrupt proton motive force by dissipating the proton concentration gradient across the cell membrane. Screening a subset of mutant strains lacking membrane-bound proteins revealed the pleiotropic effects of phenothiazines potentially rely on their ability to inhibit a wide range of critical metabolic proteins. Altogether, our study further highlights the complex roles of metabolism in persister cell formation, survival and recovery, and suggests metabolic inhibitors such as phenothiazines can be selectively detrimental to persister cells.

Solubilization of aqueous-insoluble phenothiazine drug in TX-100 micellar solution and interactions of cationic/anionic surfactants with phenothiazine–TX-100 system

This article discusses the interaction of surfactants such as tetradecyltrimethylammonium bromide (TTAB), hexadecyltrimethylammonium bromide (CTAB), cetylpyridinium chloride (CPC), cetylpyridinium bromide (CPB) and sodium dodecylsulphate (SDS) in presence of poorly soluble drug phenothiazine (PTZ) in triton X-100 (TX-100) micellar aqueous media through the investigation of different physicochemical parameters. The interaction of surfactants in PTZ–TX-100 media has been studied using conductometric technique at pH 7.4 (SP buffer) and at three temperatures. The conductometric results reveal the sequence of critical micelle concentration (CMC) values of surfactants as SDS > TTAB > CPC > CPB > CTAB. UV–vis spectroscopy studies were used to learn the interactions of surfactants with the drug PTZ in TX-100 aqueous media (at 303.15 K and pH 7.4) which suggest the interactions are highly feasible (change of Gibbs energy; ΔGb < 0) and greatly improve beyond the CMC values of corresponding surfactants. The estimated parameters using the Benesi–Hildebrand equation indicate higher binding constant (Kb) for cetylpyridinium surfactants and least for SDS and the drug–surfactant complex formations are found to be in 1:2 stoichiometric ratios. Fluorescence technique has also been employed to study the aggregation number (Nagg), Stern-Volmer constant (KSV) and micro-polarity of TTAB/CTAB/SDS in PTZ–TX-100 systems at 303.15 K. The evaluated thermodynamic parameters such as change in Gibbs energy (ΔGm0), enthalpy (ΔHm0) and entropy (ΔSm0) of micellization show the process of micellization of all studied surfactants in PTZ–TX-100 aqueous media are spontaneous and exothermic.

Visualization of the photodegradation of a therapeutic drug by chemometric-assisted fluorescence spectroscopy.

The fluorescence spectral fingerprint, also known as the excitation-emission matrix (EEM), is used to assess and visualize therapeutic drug photodegradation in combination with chemometrics. Examination of EEM-parallel factor analysis (PARAFAC) data showed that an individual component was easily separated from a mixture of photogenerated products of a heterocyclic pharmacophore, in this case, phenothiazine drugs (PTZs). Detailed investigations of both structure–EEM relationships and kinetics revealed that the components extracted from EEM–PARAFAC could be quantitatively attributed to such photogenerated products as phenothiazine sulfoxide and carbazole derivatives. EEM in combination with principal component analysis (PCA) could be used as a mapping tool to visualize information of the photodegradation process of PTZs. We also assessed the photostability of various types of PTZs containing side chains by using validated EEM–PARAFAC methodology.

Nano-titania modified cupric-based metal–organic frameworks for the on-line pass-through cleanup of phenothiazine drugs and metabolites in human plasma

SEM (A) and TEM (B) images of NTMCu-MOFs.

Transition from antagonistic to synergistic interaction in mixed micellar system by increase in alkyl chain length of alkyl trimethylammonium bromide

Herein, the electrical conductivity technique used to measure the critical micelle concentration (cmc) for pure cationic surfactants (Dodecyl/cetyltrimethylammonium bromides) and phenothiazine drug (Promethazine hydrochloride), as well as their different mole fractions in 10−3 ​mol/kg 1-methyl-3-octylimidazolium chloride (C8mim.Cl) at different temperatures. By using the cmc values the regular solution theory used to evaluate the micellar mole fraction for DTAB/CTAB-PMT mixed systems. The clint's model used to calculate the ideal critical micelle concentration (cmc∗) that helps to evaluate the ideal micellar mole fraction for studied mixed systems, both explain the deviation from ideality. In addition, interaction parameter βm helps to confirm the nature of interaction (either antagonistic or synergistic) for the studied systems. The calculated parameters confirm a transition in the binding nature from antagonistic to synergistic with the increase in alkyl chain length of cationic surfactant i.e., component of the binary mixed system. The ease of micellization for the studied systems discussed by the standard Gibb's energy of micellization (ΔGm0), as well as the standard enthalpy and standard entropy of micellization were (ΔHm0) and (ΔSm0), respectively to discuss the stability of the studied systems.

Impact of numerous media on association, interfacial, and thermodynamic properties of promethazine hydrochloride (PMT) + benzethonium chloride (BTC) mixture of various composition

Herein, the impact of numerous media of fixed concentration (H2O, NaCl (50 mmol·kg−1), urea (U, 300 mmol·kg−1) and thiourea (TU, 300 mmol·kg−1)) on the association, interfacial, and thermodynamic properties of promethazine hydrochloride (PMT) and benzethonium chloride (BTC, cationic antimicrobial surfactant)) mixture in several compositions were examined via tensiometry method at room temperature (298.15 K). Along with tensiometry, the interaction between PMT and BTC was also assessed by applying the UV–visible as well as FTIR spectroscopy in an aqueous solution. PMT is used for the symptomatic relief of various allergic conditions. The employed systems critical micelle concentrations (cmc) value was acquired much lower than their calculated corresponding ideal cmc value (cmcid) owing to the interaction between components (PMT and BTC) and the interaction amongst PMT and BTC further increases through increase in mole fraction (α1) of first component (BTC). Diverse solution and surface parameters, thermodynamic parameters, etc. have been processed by use of several purported theoretical models. In NaCl media, the cmc value of singular along with mixed species (PMT + BTC) of each composition were found fewer in magnitude than aqueous media while magnitude increased in U/TU media and TU is more valuable in boosting the cmc than U. Activity coefficient value in each case was continuously found below one due to attractive interaction amongst PMT and BTC. The attained Γmax and Amin value of PMT + BTC implies that the formed mixed interface is the closer packing of PMT and BTC. Excess free energy (ΔGex) was attained negative, exhibiting that the mixed micellar/mixed monolayer stability was higher with the singular component’s micelles/monolayer. The shift in frequency of pure component were attained by FT-IR study by addition of other component that also signifies the straightforward interaction among ingredients. Existing research outcomes revealed a straightforward methodology to design the employed surfactant as an efficient ingredient as a drug delivery vehicle for phenothiazine drug.

Aggregational behaviour of promethazine hydrochloride and TX-45 surfactant mixtures: A multi-techniques approach

Herein, a multi-techniques approach of aggregation of promethazine hydrochloride (PMT, phenothiazine drug) and TX-45 surfactant (nonionic) mixtures have been explored thoroughly. Distinct media (H2O/0.05 mol kg−1 NaCl/0.50 mol kg−1 urea (U) /0.50 mol kg−1 thiourea (TU)) were used for tensiometric measurement of pure and mixed system (PMT + TX-45). A UV–visible and FT-IR studies were employed to explore the interaction between drug and TX-45 in an aqueous system. The achieved results of critical micelles concentration (cmc) of PMT + TX-45 from tensiometric measurement was found large deviation their corresponding ideal value (cmcid) which revealed the nonideal behaviour of mixed system (PMT + TX-45). In NaCl media, the cmc value was attained lesser than aqueous solution, confirming that a strong interaction amongst PMT and TX-45 in NaCl whereas in the existence of U/TU, interaction between components decreased as compared with aqueous system that's why cmc value increased. Composition of the component in mixed micelles and mixed interface was attained more for TX-45 than PMT in all solvents. Mixed micelles along with interface interaction parameters showed the attractive/synergism among both employed components. Activity coefficient (solution and interface) for PMT and TX-45 was attained less than one depicting the non-ideal as well as attractive interaction amongst components. Several assessed thermodynamic parameters showed that mixed micellization, as well as mixed interface formed by PMT + TX-45 mixture, is feasible as well as spontaneous, and feasibility increased in NaCl media, decreases in U/TU solvent as compared with aqueous media. UV–visible study showed fine interaction of PMT with added TX-45. FT-IR investigation showed that the shifting in wavelength frequency of singular component in presence of other component elucidate the interaction among them.

Phenothiazines as dual inhibitors of SARS-CoV-2 main protease and COVID-19 inflammation

COVID-19, caused by the severe acute respiratory coronavirus 2 (SARS-CoV-2), currently has no treatment for acute infection. The main protease (Mpro) of SARS-CoV-2 is an essential enzyme for viral replication and an attractive target for disease intervention. The phenothiazine moiety has demonstrated drug versatility for biological systems, including inhibition of butyrylcholinesterase, a property important in the cholinesterase anti-inflammatory cascade. Nineteen phenothiazine drugs were investigated using in silico modelling techniques to predict binding energies and inhibition constants (Ki values) with SARS-CoV-2 Mpro. Because most side-effects of phenothiazines are due to interactions with various neurotransmitter receptors and transporters, phenothiazines with few such interactions were also investigated. All compounds were found to bind to the active site of SARS-CoV-2 Mpro and showed Ki values ranging from 1.30 to 52.4 µM in a rigid active site. Nine phenothiazines showed inhibition constants &lt;10 µM. The compounds with limited interactions with neurotransmitter receptors and transporters showed micromolar (µM) Ki values. Docking results were compared with remdesivir and showed similar interactions with key residues Glu-166 and Gln-189 in the active site. This work has identified several phenothiazines with limited neurotransmitter receptor and transporter interactions and that may provide the dual action of inhibiting SARS-CoV-2 Mpro to prevent viral replication and promote the release of anti-inflammatory cytokines to curb viral-induced inflammation. These compounds are promising candidates for further investigation against SARS-CoV-2.

Antipsychotic phenothiazine drugs bind to KRAS in vitro.

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.

Chlorpromazine and promethazine reduces Brain injury through RIP1-RIP3 regulated activation of NLRP3 inflammasome following ischemic stroke

ABSTRACT Objectives: Stroke is an important cause of death and disability. Recent evidence suggests that post-stroke inflammation is an important factor in stroke pathology and a root cause of its lasting consequences. Phenothiazine drugs, like chlorpromazine and promethazine (C + P), induce hypothermia and have been shown to play a major role in neuroprotection. In the present study, we investigated this neuroprotective mechanism by assessing the anti-inflammatory effect of these drugs. Methods: Adult Sprague-Dawley rats underwent 2 h of middle cerebral artery occlusion (MCAO) followed by 6 or 24 h of reperfusion, with or without C + P (8 mg/kg). Infarct volumes, neurological deficits, along with mRNA and protein quantities of receptor-interacting protein 1 (RIP1), receptor-interacting protein 3 (RIP3), NLRPyrin domain containing 3 (NLRP3), and interleukin-1β (IL-1β) were assessed, as well as the infiltration of neutrophils and macrophages. Results: C + P induced hypothermia that significantly reduced RIP1, RIP3, NLRP3 and IL-1β expression, infarction, and immune cell infiltration, while C + P treatment with temperature control at 37°C induced lesser effect. Conclusion: These findings suggest that the anti-inflammatory effect of C + P may be dependent on drug-induced hypothermia and regulation of the NLRP3 inflammasome via the RIP1/RIP3 complex. Future investigations are needed regarding C + P as potential treatment of ischemic stroke.