Application of electron paramagnetic resonance spectroscopy for determining the relative nanoenvironment fluidity of polymeric micelles.

Polymeric micelles are nanocarriers for drug, protein and gene delivery due to their unique core/shell structure, which encapsulates and protects therapeutic cargos with diverse physicochemical properties. However, information regarding the micellar nanoenvironment's fluidity can provide unique insight into their makeup. In this study, we used electron paramagnetic resonance (EPR) spectroscopy to study free radical spin probe (5-doxylstearate methyl ester, 5-MDS, and 16-doxylstearic acid, 16-DS) behaviour in methoxy-poly(ethylene oxide)-poly(α-benzyl carboxylate-ε-caprolactone) (PEO-PBCL) and methoxy-poly(ethylene oxide)-poly(ε-caprolactone) (PEO-PCL) polymeric micelles. Spin probes provided information about the spectroscopic rotational correlation time (τ, s) and the spectroscopic partition parameter F. We hypothesized that spin probes would partition into the polymeric micelles, and these parameters would be calculated. The results showed that both 5-MDS and 16-DS spectra were modulated in the presence of polymeric micelles. Based on τ values, 5-MDS revealed that PEO-PCL (τ = 3.92 ± 0.26 × 10-8 s) was more fluid than PEO-PBCL (τ = 7.15 ± 0.63 × 10-8 s). The F parameter, however, could not be calculated due to the rotational hindrance of the probe within the micelles. With 16-DS, more probe rotation was observed, and although the F parameter could be calculated, it was not helpful to distinguish the micelles' fluidity. Also, doxorubicin-loading interfered with the spin probes, particularly for 16-DS. However, using simulations, we could distinguish the hydrophilic and hydrophobic components of the 16-DS probe. The findings suggest that EPR spectroscopy is a valuable method for determining core fluidity in polymeric micelles.

Comparing the oxidative functions of neutrophil myeloperoxidase and cytochrome P450 enzymes in drug metabolism.

Drug metabolism is an essential process that chemically alters xenobiotic substrates to activate or terminate drug activity. Myeloperoxidase (MPO) is a neutrophil-derived haem-containing enzyme that is involved in killing invading pathogens, although consequentially, this same oxidative activity can produce metabolites that damage host tissue and play a role in various human pathologies. Cytochrome P450s (CYPs) are a superfamily of haem-containing enzymes that are significantly involved in the metabolism of drugs by functioning as monooxygenases and can be induced or inhibited, resulting in significant drug-drug interactions that lead to unanticipated adverse drug reactions. In this review, the functions of drug metabolism of MPO and CYPs are explored, along with their involvement and association for common enzymatic pathways by certain xenobiotics. MPO and CYPs metabolize numerous xenobiotics, although few reported studies have made a direct comparison between both enzymes. Additionally, we employed molecular docking to compare the active site and haem prosthetic group of MPO and CYPs, supporting their similar catalytic activities. Furthermore, we performed LCMS analysis and observed a shared hydroxylated mefenamic acid metabolite produced in both enzymatic systems. A proper understanding of the enzymology and mechanisms of action of MPO and CYPs is of significant importance when enhancing the beneficial functions of drugs in health and diminishing their damaging effects on diseases. Therefore, awareness of drugs and xenobiotic substrates involved in MPO and CYPs metabolism pathways will add to the knowledge base to foresee and prevent potential drug interactions and adverse events.

Proof of concept: Pull down assay using bovine serum albumin and an immunomodulator small molecule.

In the past decade, there has been increasing interest in use of small molecules for immunomodulation. The affinity-based pull-down purification is an essential tool for target identification of small molecules and drug discovery. This study presents our recent efforts to investigate the cellular target(s) of Compound A, a small molecule with demonstrated immunomodulatory properties in human peripheral blood mononuclear cells (PBMCs). While we have previously observed the immunomodulatory activity of Compound A in PBMCs, the specific molecular targets underlying its effects remains elusive. To address this challenge, we synthesized a trifluoromethyl phenyl diazirine (TPD)-bearing trifunctional Probe 1 based on the chemical structure of Compound A, which could be used in a pull-down assay to efficiently bind to putative cellular targets via photoaffinity labelling. In this report, we utilized bovine serum albumin (BSA) as a model protein to establish a proof-of-concept in order to assess the suitability of Probe 1 for binding to an endogenous target. By the successful synthesis of Probe 1 and demonstrating the efficient binding of Probe 1 to BSA, we propose that this method can be used as a tool for further identification of potential protein targets of small molecules in living cells. Our findings provide a valuable starting point for further investigations into the molecular mechanisms underlying the immunomodulatory effects of Compound A.

Target identification of small molecules: an overview of the current applications in drug discovery.

Target identification is an essential part of the drug discovery and development process, and its efficacy plays a crucial role in the success of any given therapy. Although protein target identification research can be challenging, two main approaches can help researchers make significant discoveries: affinity-based pull-down and label-free methods. Affinity-based pull-down methods use small molecules conjugated with tags to selectively isolate target proteins, while label-free methods utilize small molecules in their natural state to identify targets. Target identification strategy selection is essential to the success of any drug discovery process and must be carefully considered when determining how to best pursue a specific project. This paper provides an overview of the current target identification approaches in drug discovery related to experimental biological assays, focusing primarily on affinity-based pull-down and label-free approaches, and discusses their main limitations and advantages.

Effect of Plasma-Activated Water Bubbles on Fusarium graminearum, Deoxynivalenol, and Germination of Naturally Infected Barley during Steeping.

Contamination of barley by deoxynivalenol (DON), a mycotoxin produced by Fusarium graminearum, causes considerable financial loss to the grain and malting industries. In this study, two atmospheric cold plasma (ACP) reactors were used to produce plasma-activated water (PAW) bubbles. The potential of PAW bubbles for the steeping of naturally infected barley (NIB) during the malting process was investigated. The PAW bubbles produced by treating water for 30 min using a bubble spark discharge (BSD) at low temperature resulted in the greatest concentration of oxygen-nitrogen reactive species (RONS). This treatment resulted in 57.3% DON degradation compared with 36.9% in the control sample; however, the same treatment reduced germination significantly (p < 0.05). Direct BSD ACP treatment for 20 min at low temperature and indirect treatment for 30 min increased the percentage of germinated rootlets of the seedlings compared with the control. Considering both the DON reduction and germination improvement of barley seeds, continuous jet ACP treatment for 30 min performed better than the other treatments used in this study. At higher temperature of PAW bubbles, the concentration of RONS was significantly (p < 0.05) reduced. Based on quantitative polymerase chain reaction (qPCR) analysis and fungal culture tests, the PAW bubble treatment did not significantly reduce infection of NIB. Nonetheless, this study provides useful information for the malting industry for PAW treatment optimization and its use in barley steeping for DON reduction and germination improvement.

Heating of consumer cannabis oils can lead to free radical initiated degradation, causing CBD and THC depletion.

Commercial cannabis oil products are widely available in Canada even though there is a significant gap in scientific information regarding them. Oils, such as vegetable oils, are known to undergo oxidative changes through free radical mechanisms when they are heated or aged, but the cannabis oils used in this study did not have expiry dates or best-before usage dates. This led to the question of how these products would be affected with time. We hypothesized that cannabis oils would produce increased concentrations of free radicals in aging-simulated conditions, which would be related to a decrease in cannabidiol (CBD) or Δ9-tetrahydrocannabinol (THC) content. Cannabis oils and their respective vehicles (oils) were heated using two protocols: One (moderate aging method) used a 2-day heating protocol at 50 °C, and the other (enhanced aging method) used a 14-day heating protocol at 70 °C. We used electron paramagnetic resonance (EPR) spectroscopy for free radical analysis using the spin trapping technique using 200 mM PBN and 0.02 mM CuCl2 (for peroxide breakdown to free radicals). For active ingredient analysis (CBD, THC), we used LC/MS. Cannabis oils that contained unsaturated oils as their vehicles, such as olive or sunflower oil, all showed varying degrees of free radical formation. In both aged and unaged oils containing CBD or THC, less free radical formation was detected compared to the vehicle controls. Cannabis oils using medium-chain triglycerides (MCT) showed little or no free radical formation. The most significant decrease in CBD or THC was observed in the products using sunflower oil, to a lesser extent in MCT oil, and THC also decreased in olive oil. These findings are important for consumers and policymakers considering using such products in hot beverages or cooking and highlighting the importance of appropriate storage conditions.

Neutrophil Myeloperoxidase-Mediated N-Demethylation of Quetiapine Leads to N-Desalkylquetiapine, a Pharmacologically Active Cytochrome P450 Metabolite.

The atypical antipsychotic drugs, quetiapine and clozapine, are associated with idiosyncratic drug reactions (such as agranulocytosis or neutropenia) that are thought to involve reactive metabolites. Neutrophil myeloperoxidase (MPO) metabolism of quetiapine is not well-studied, but is metabolized by cytochrome P450. Based on structural similarity to clozapine, we hypothesized that quetiapine can be metabolized by MPO and that there is overlap between cytochrome P450 and MPO metabolism of quetiapine. The interaction of quetiapine and clozapine with MPO and MPO chlorination activity was studied using UV-vis spectrophotometry. The metabolites were characterized using liquid chromatography-mass spectrometry (LC-MS), and electron paramagnetic resonance (EPR) spectroscopy was used for detecting drug-catalyzed glutathione oxidation. In the presence of quetiapine, MPO compound II accumulated for about 7.5 min, whereas in the presence of clozapine, MPO compound II was not observed as it was rapidly reduced back to the resting state. Increasing quetiapine concentrations resulted in a decrease in MPO chlorination activity, while the opposite result was found in the case of clozapine. UV-vis spectral studies showed no change when quetiapine was oxidized in the absence and presence of chloride anion (Cl-, to catalyze chlorination reactions). Significant changes, however, were observed in the same assay with clozapine, where Cl- appeared to hinder the rate of clozapine metabolism. The MPO-catalyzed hydroxylated and dealkylated metabolites of quetiapine and hydroxylated metabolites of clozapine were observed from the LC-MS analyses, particularly when Cl- was included in the reaction. In addition, hydroxylated, dealkylated, and a proposed sulfoxide metabolite of quetiapine were also observed in the reaction catalyzed by human microsomes/NADPH. Lastly, compared to quetiapine, clozapine metabolism by MPO/H2O2 and glutathione produced more glutathionyl radicals using EPR spin trapping. In conclusion, MPO/H2O2/Cl- was shown to metabolize quetiapine to S-oxidation and P450-like dealkylation products, and quetiapine metabolites were generally less reactive than clozapine.

ß-Caryophyllene Induces Apoptosis and Inhibits Angiogenesis in Colorectal Cancer Models.

Beta-Caryophyllene (BCP), a naturally occurring sesquiterpene abundantly found in cloves, hops, and cannabis, is the active candidate of a relatively new group of vascular-inhibiting compounds that aim to block existing tumor blood vessels. Previously, we have reported the anti-cancer properties of BCP by utilizing a series of in-vitro anti-tumor-related assays using human colorectal carcinoma cells. The present study aimed to investigate the effects of BCP on in-vitro, ex-vivo, and in-vivo models of anti-angiogenic assays and evaluate its anti-cancer activity in xenograft tumor (both ectopic and orthotopic) mice models of human colorectal cancer. Computational structural analysis and an apoptosis antibody array were also performed to understand the molecular players underlying this effect. BCP exhibited strong anti-angiogenic activity by blocking the migration of endothelial cells, tube-like network formation, suppression of vascular endothelial growth factor (VEGF) secretion from human umbilical vein endothelial cells and sprouting of rat aorta microvessels. BCP has a probable binding at Site#0 on the surface of VEGFR2. Moreover, BCP significantly deformed the vascularization architecture compared to the negative control in a chick embryo chorioallantoic membrane assay. BCP showed a remarkable reduction in tumor size and fluorescence molecular tomography signal intensity in all the mice treated with BCP, in a dose-dependent relationship, in ectopic and orthotopic tumor xenograft models, respectively. The histological analysis of the tumor from BCP-treated mice revealed a clear reduction of the density of vascularization. In addition, BCP induced apoptosis through downregulation of HSP60, HTRA, survivin, and XIAP, along with the upregulation of p21 expressions. These results suggest that BCP acts at multiple stages of angiogenesis and could be used as a promising therapeutic candidate to halt the growth of colorectal tumor cells.

The many roles of myeloperoxidase: From inflammation and immunity to biomarkers, drug metabolism and drug discovery.

This review provides a practical guide to myeloperoxidase (MPO) and presents to the reader the diversity of its presence in biology. The review provides a historical background, from peroxidase activity to the discovery of MPO, to its role in disease and drug development. MPO is discussed in terms of its necessity, as specific individuals lack MPO expression. An underlying theme presented throughout brings up the question of the benefit and burden of MPO activity. Enzyme structure is discussed, including accurate masses and glycosylation sites. The catalytic cycle of MPO and its corresponding pathways are presented, with a discussion of the importance of the redox couples of the different states of MPO. Cell lines expressing MPO are discussed and practically summarized for the reader, and locations of MPO (primary and secondary) are provided. Useful methods of MPO detection are discussed, and how these can be used for studying disease processes are implied through the presentation of MPO as a biomarker. The presence of MPO in neutrophil extracellular traps is presented, and the activators of the former are provided. Lastly, the transition from drug metabolism to a target for drug development is where the review concludes.

Interactions of the antioxidant enzymes NAD(P)H: Quinone oxidoreductase 1 (NQO1) and NRH: Quinone oxidoreductase 2 (NQO2) with pharmacological agents, endogenous biochemicals and environmental contaminants.

NAD(P)H: Quinone Oxidoreductase 1 (NQO1) is an antioxidant enzyme that catalyzes the two-electron reduction of several different classes of quinone-like compounds (quinones, quinone imines, nitroaromatics, and azo dyes). One-electron reduction of quinone or quinone-like metabolites is considered to generate semiquinones to initiate redox cycling that is responsible for the generation of reactive oxygen species and oxidative stress and may contribute to the initiation of adverse drug reactions and adverse health effects. On the other hand, the two-electron reduction of quinoid compounds appears important for drug activation (bioreductive activation) via chemical rearrangement or autoxidation. Two-electron reduction decreases quinone levels and opportunities for the generation of reactive species that can deplete intracellular thiol pools. Also, studies have shown that induction or depletion (knockout) of NQO1 were associated with decreased or increased susceptibilities to oxidative stress, respectively. Moreover, another member of the quinone reductase family, NRH: Quinone Oxidoreductase 2 (NQO2), has a significant functional and structural similarity with NQO1. The activity of both antioxidant enzymes, NQO1 and NQO2, becomes critically important when other detoxification pathways are exhausted. Therefore, this article summarizes the interactions of NQO1 and NQO2 with different pharmacological agents, endogenous biochemicals, and environmental contaminants that would be useful in the development of therapeutic approaches to reduce the adverse drug reactions as well as protection against quinone-induced oxidative damage. Also, future directions and areas of further study for NQO1 and NQO2 are discussed.

Reversing T-cell exhaustion in immunotherapy: a review on current approaches and limitations.

Introduction:T cell functions are altered during chronic viral infections and tumor development. This is mainly manifested by significant changes in T cells' epigenetic and metabolic landscapes, pushing them into an 'exhausted' state. Reversing this T cell exhaustion has been emerging as a 'game-changing' therapeutic approach against cancer and chronic viral infection.Areas covered:This review discusses the cellular pathways related to T cell exhaustion, and the clinical development and possible cellular targets that can be exploited therapeutically to reverse this exhaustion. We searched various databases (e.g. Google Scholar, PubMed, Elsevier, and other scientific database sites) using the keywords T cell exhaustion, T cell activation, co-inhibitory receptors, and reversing T cell exhaustion.Expert opinion:The discovery of the immune checkpoints pathways represents a significant milestone toward understanding and reversing T cell exhaustion. Antibodies that target these pathways have already demonstrated promising activities in reversing T cell exhaustion. Nevertheless, there are still many associated limitations. In this context, next-generation alternatives are on the horizon. This includes the use of small molecules to block the immune checkpoints' receptors, combining them with other treatments, and identifying novel, safer and more effective immunotherapeutic targets.

Tackling Immune Targets for Breast Cancer: Beyond PD-1/PD-L1 Axis.

The burden of breast cancer is imposing a huge global problem. Drug discovery research and novel approaches to treat breast cancer have been carried out extensively over the last decades. Although immune checkpoint inhibitors are showing promising preclinical and clinical results in treating breast cancer, they are facing multiple limitations. From an immunological perspective, a recent report highlighted breast cancer as an "inflamed tumor" with an immunosuppressive microenvironment. Consequently, researchers have been focusing on identifying novel immunological targets that can tune up the tumor immune microenvironment. In this context, several novel non-classical immune targets have been targeted to determine their ability to uncouple immunoregulatory pathways at play in the tumor microenvironment. This article will highlight strategies designed to increase the immunogenicity of the breast tumor microenvironment. It also addresses the latest studies on targets which can enhance immune responses to breast cancer and discusses examples of preclinical and clinical trial landscapes that utilize these targets.

Treatment of a mixture of pharmaceuticals, herbicides and perfluorinated compounds by powdered activated carbon and ozone: Synergy, catalysis and insights into non-free OH contingent mechanisms.

Powdered activated carbon (PAC) is a strong adsorbent also capable of catalyzing ozonation processes. Ozone (O3) and PAC were simultaneously applied to treat a mixture of 17 micropollutants (MPs) at low concentrations, including 13 compounds that were studied for the first time by simultaneous addition of O3 and PAC system. Synergy and catalysis improved the removals and specific degradation rates of MPs in the first minute of the treatment. Radical probing experiments showed that scavenging hydroxyl radicals (OH) did not have a significant impact on the removals, while scavenging other reactive oxygen species was more influential. A detailed study by electron paramagnetic resonance spectroscopy ascertained that the decomposition of ozone in presence of PAC at neutral pH did not boost the generation of free OH. Instead, adsorbed OH was likely produced as PAC-HO along with other oxidizing species resulting from adsorbed singlet oxygen and superoxide radicals.

Isoniazid and host immune system interactions: A proposal for a novel comprehensive mode of action.

The known mode of action of isoniazid (INH) is to inhibit bacterial cell wall synthesis following activation by the bacterial catalase-peroxidase enzyme KatG in Mycobacterium tuberculosis (Mtb). This simplistic model fails to explain (a) how isoniazid penetrates waxy granulomas with its very low lipophilicity, (b) how isoniazid kills latent Mtb lacking a typical cell wall, and (c) why isoniazid treatment time is remarkably long in contrast to most other antibiotics. To address these questions, a novel comprehensive mode of action of isoniazid has been proposed here. Briefly, isoniazid eradicates latent tuberculosis (TB) by prompting slow differentiation of pro-inflammatory monocytes and providing protection against reactive species-induced "self-necrosis" of phagocytes. In the case of active TB, different immune cells form INH-NAD+ adducts to inhibit Mtb's cell wall biosynthesis. This additionally suggests that the antibacterial properties of INH do not rely on KatG of Mtb. As such, isoniazid-resistant TB needs to be re-evaluated.

Comprehensive in vitro characterization of PD-L1 small molecule inhibitors.

Blockade of the programmed cell death 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) interaction has emerged as a powerful strategy in cancer immunotherapy. Recently, there have been enormous efforts to develop potent PD-1/PD-L1 inhibitors. In particular, Bristol-Myers Squibb (BMS) and Aurigene Discovery Technologies have individually disclosed several promising PD-1/PD-L1 inhibitors, whose detailed experimental data are not publicly disclosed. In this work, we report the rigorous and systematic in vitro characterization of a selected set of potent PD-1/PD-L1 macrocyclic peptide (BMSpep-57) and small-molecule inhibitors (BMS-103, BMS-142) from BMS and a peptidomimetic small-molecule inhibitor from Aurigene (Aurigene-1) using a series of biochemical and cell-based assays. Our results confirm that BMS-103 and BMS-142 are strongly active in biochemical assays; however, their acute cytotoxicity greatly compromised their immunological activity. On the other hand, Aurigene-1 did not show any activity in both biochemical and immunological assays. Furthermore, we also report the discovery of a small-molecule immune modulator, whose mode-of-action is not clear; however, it exhibits favorable drug-like properties and strong immunological activity. We hope that the results presented here will be useful in guiding the development of next-generation PD-1/PD-L1 small molecule inhibitors.

Myeloperoxidase-mediated oxidation of edaravone produces an apparent non-toxic free radical metabolite and modulates hydrogen peroxide-mediated cytotoxicity in HL-60 cells.

Edaravone is considered to be a potent antioxidant drug known to scavenge free radical species and prevent free radical-induced lipid peroxidation. In this study, we investigated the effect of edaravone on the myeloperoxidase (MPO) activity, an enzyme responsible for the production of an array of neutrophil-derived oxidants that can cause cellular damage. The addition of edaravone to the reaction of MPO and hydrogen peroxide (H2O2) significantly enhanced the reduction of MPO Compound II back to native MPO. Interestingly, the MPO-mediated production of toxic hypochlorous acid exhibited a concentration-dependent biphasic effect, with the apparent optimal edaravone concentration at 10 µM. Oxidation of edaravone by MPO was examined by various analytical methods. An MPO-catalyzed product(s) of edaravone was identified at 350 nm by kinetic analysis of UV-Vis spectroscopy. Several MPO-catalyzed metabolites of edaravone were proposed from the LC-MS analyses, including oxidized dimers from edaravone radicals. Electron spin resonance (ESR) spin trapping detected a carbon-centred radical metabolite of edaravone. NMR studies revealed that there are two exchangeable hydrogens, one of which is on the α-carbon, justifying the carbon-centred edaravone radical produced from MPO. Despite the formation of an edaravone carbon-radical metabolite, it did not appear to effectively oxidize GSH (in comparison with phenoxyl radicals). Viability (ATP) and cytotoxicity (LDH release) assays showed a concentration-dependent effect of edaravone on HL-60 cells treated with either a bolus concentration of 30 µM H2O2 or a flux of H2O2 generated by 5 mM glucose and 10 mU/mL glucose oxidase. The H2O2-induced toxicity was ameliorated at high edaravone concentrations (100-200 µM). In contrast, low concentrations of edaravone (1-10 µM) exacerbated the H2O2-induced toxicity. However, the effect of edaravone at low concentration (0-10 µM) appeared more prominent with the LDH assay only. The cellular findings correlated with the biochemical studies with respect to hypochlorous acid formation. These findings provide interesting perspectives regarding the duality of edaravone as an antioxidant drug.

Caution for the routine use of phenol red - It is more than just a pH indicator.

Phenol red (PR) is the standard pH indicator in various cell and tissue culture media, as it provides a quick check for the health of the culture. PR has also been used in multiple protocols to detect cellular hydrogen peroxide as well as peroxidase activity from human peroxidase enzymes. The majority of promyelocytic leukemia cell lines (e.g. HL-60 cells) express myeloperoxidase (MPO), which may react with PR, especially as the latter is present in cell culture media at sufficient concentrations (~15 µM) to partake in redox reactions. Moreover, phenolic molecules are often efficient donor substrates for peroxidase enzymes. In this study, we hypothesized that MPO metabolism of PR via MPO-expressing HL-60 cells could result in PR metabolite(s) that could modulate cell viability. We used purified human MPO for UV-visible spectrophotometry, electron paramagnetic resonance (EPR) and LC-MS analyses to investigate PR peroxidation. 2-chloro-5,5-dimethyl-1,3-cyclohexanedione (monochloro-dimedone, MCD) was used to assess the effect of PR on MPO-catalyzed chlorination activity, and we assessed PR uptake by HL-60 cells using LC-MS analysis. Lastly, we investigated the impact of PR metabolism by intracellular MPO on cell viability (ATP, using CellTiter-Glo®), cytotoxicity (using trypan blue), and on reduced and oxidized glutathione (using GSH/GSSG-Glo™). Our results demonstrate that PR undergoes oxidative halogenation via MPO, resulting in its UV-vis spectral changes due to the formation of mono- and di-halogenated products. Moreover, a significant increase in MPO-catalyzed chlorination of MCD and an increase in glutathionyl radical detection (using EPR) were observed in the presence of PR. Our in-vitro studies revealed that PR is readily taken up by HL-60 cells and its metabolism by intracellular MPO leads to a significant decrease in cellular glutathione as well as a significant increase in glutathione disulphide formation. In spite of the latter, PR had no considerable effect on HL-60 cell viability. These results provide evidence that while no overt decrease in cell viability may be observed, PR does impart redox activity, which investigators should be wary of in experimental protocols.

Eosinophil peroxidase oxidizes isoniazid to form the active metabolite against M. tuberculosis, isoniazid-NAD.

The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD+) by the mycobacterial catalase-peroxidase enzyme, KatG, was known to be the major component of the mode of action of isoniazid (INH), an anti-tuberculosis drug. However, there are other enzymes that may catalyze this reaction. We have previously reported that neutrophil myeloperoxidase (MPO) is capable of metabolizing INH through the formation of INH-NAD+ adduct, which could be attributed to being a possible mode of action of INH. However, eosinophilic infiltration of the lungs is more pronounced and characteristic of granulomas in Mycobacterium tuberculosis-infected patients. Thus, the aim of the present study is to investigate the role of eosinophil peroxidase (EPO), a key eosinophil enzyme, during INH metabolism and the formation of its active metabolite, INH-NAD+ using purified EPO and eosinophils isolated from asthmatic donors. UV-Vis spectroscopy revealed INH oxidation by EPO led to a new product (λmax = 326 nm) in the presence of NAD+. This adduct was confirmed to be INH-NAD+ using LC-MS analysis where the intact adduct was detected (m/z = 769). Furthermore, EPO catalyzed the oxidation of INH and formed several free radical intermediates as assessed by electron paramagnetic resonance (EPR) spin-trapping; a carbon-centred radical, which is considered to be the reactive metabolite that binds with NAD+, was found when superoxide dismutase was included in the reaction. Our findings suggest that eosinophilic EPO may also play a role in the pharmacological activity of INH through the formation of INH-NAD+ adduct, and supports further evidence that human cells and enzymes are capable of producing the active metabolite involved in tuberculosis treatment.

Isoniazid induces a monocytic-like phenotype in HL-60 cells.

Isoniazid (INH) is one of the oldest drugs for the treatment of tuberculosis (TB) and is of continual clinical and research interest. The aim of the current study is to investigate the ability of INH to induce monocyte differentiation and the underlying signaling pathway involved in this phenomenon using HL-60 cells. In this study, HL-60 cells were treated with different non-cytotoxic concentrations of INH or vitamin D (a well-known inducer of monocytic differentiation) to determine key functional changes in the phenotype of these cells using several biochemical and cytobiological experiments. HL-60 cells are derived from human promyelocytic leukemia and bear some resemblance to promyelocytes, which differentiate into various cell types. INH-induced differentiation was confirmed to occur in a concentration-dependent manner through several functional markers such as nonspecific esterase activity, NADPH oxidase activity and expression of surface markers CD14 and CD16 (characteristic of monocytes). INH-induced monocytic-like differentiation in HL-60 cells and demonstrated that at least 25% of cells were differentiated within the range of the pharmacological concentrations of INH. To determine the effects of INH on HL-60 cells, we applied quantitative proteomics that revealed 32 proteins were altered significantly in pathways that could involve differentiation signals. Lastly, INH activated the ERK-1/MAPK signaling pathway based on detection of phosphorylated ERK-1. These in vitro findings in HL-60 cells warrant further study using promyelocytes or hematopoietic stem cells to evaluate the physiological capability of INH to induce monocytic differentiation that may aid in host defense against TB.