Metadynamics Study of Lipid-Mediated Antibacterial Toxin Binding to the EmrE Multiefflux Protein.

EmrE is a bacterial efflux protein in the small multidrug-resistant (SMR) family present in Escherichia coli. Due to its small size, 110 residues in each dimer subunit, it is an ideal model system to study ligand-protein-membrane interactions. Here in our work, we have calculated the free energy landscape of benzyltrimetylammonium (BTMA) and tetraphenyl phosphonium (TPP) binding to EmrE using the enhanced sampling method-multiple walker metadynamics. We estimate that the free energy of BTMA binding to EmrE is -21.2 ± 3.3 kJ/mol and for TPP is -43.6 ± 3.8 kJ/mol. BTMA passes through two metastable states to reach the binding pocket, while TPP has a more complex binding landscape with four metastable states and one main binding site. Our simulations show that the ligands interact with the membrane lipids at a distance 1 nm away from the binding site which forms a broad local minimum, consistent for both BTMA and TPP. This site can be an alternate entry point for ligands to partition from the membrane into the protein, especially for bulky and/or branched ligands. We also observed the membrane lipid and C-terminal 110HisA form salt-bridge interactions with the helix-1 residue 22LysB. Our free energy estimates and clusters are in close agreement with experimental data and give us an atomistic view of the ligand-protein-lipid interactions. Understanding the binding pathway of these ligands can guide us in future design of ligands that can alter or halt the function of EmrE.

Achieving property enhancements in dental resin composites via reduced concentrations of camphorquinone within a ternary initiator system.

OBJECTIVES: The study aimed to assess the impact of diphenyliodonium hexafluorophosphate (DPI) on the physicochemical properties of experimental resin composites (ECRs) featuring reduced concentrations of camphorquinone (CQ)/amine. METHODS: Five concentrations of CQ (0.125, 0.25, 0.5, 0.75, and 1 mol%) with dimethylaminoethyl amine benzoate (EDAB) in a 1:2 mol% ratio (CQ:EDAB) were incorporated into a 50:50 mass% monomer blend of bisphenol glycidyl methacrylate (BisGMA) and triethyleneglycol dimethacrylate (TEGDMA). An additional 5 groups with the same CQ:EDAB concentrations had 0.5 mol% DPI added. Each resin group contained 60 wt% of 0.7 µm barium-alumino-silicate glass. Light transmission (n = 3), real-time degree of polymerization (n = 3), temperature change during polymerization (n = 5), polymerization shrinkage strain (n = 3), flexural strength, and modulus (n = 12), as well as water sorption and solubility (n = 5), were evaluated. Data were analyzed using two-way ANOVA and Tukey's post-hoc test (α = 0.05). RESULTS: Light transmission was reduced in groups containing 0.125 and 0.25 mol% of CQ without DPI. DPI increased temperature, degree and rate of polymerization, despite the reduction in CQ/amine concentration. Additionally, there was an increase in polymerization shrinkage strain, flexural strength and modulus, and a reduction in water sorption and solubility in ECRs with DPI, even with lower concentrations of CQ/EDAB. SIGNIFICANCE: DPI improved the assessed properties of composites across various concentrations of CQ/EDAB, showing the benefit of reducing the quantity of CQ used without compromising the properties and curing of the resin composites.

Diphenylene Iodonium as a Prominent Halogen Bond Donor: The Case of Human Monoamine Oxidase B.

Herein we have investigated the formation and interplay of several noncovalent interactions (NCIs) involved in the inhibition of human monoamine oxidase B (MAO B). Concretely, an inspection of the Protein Data Bank (PDB) revealed the formation of a halogen bond (HlgB) between a diphenylene iodonium (DPI) inhibitor and a water molecule present in the active site, in addition to a noncovalent network of interactions (e. g. lone pair-π, hydrogen bonding, OH-π, CH-π and π-stacking interactions) with surrounding protein residues. Several theoretical models were built to understand the strength and directionality features of the HlgB in addition to the interplay with other NCIs present in the active site of the enzyme. Besides, a computational study was carried out using DPI as HlgB donor and several electron rich molecules (CO, H2O, CH2O, HCN, pyridine, OCN-, SCN-, Cl- and Br-) as HlgB acceptors. The results were analyzed using several state-of-the-art computational tools. We expect that our results will be useful for those scientists working in the fields of rational drug design, chemical biology as well as supramolecular chemistry.

Investigation of the Dysfunction Caused by High Glucose, Advanced Glycation End Products, and Interleukin-1 Beta and the Effects of Therapeutic Agents on the Microphysiological Artery Model.

Diabetes mellitus (DM) has substantial global implications and contributes to vascular inflammation and the onset of atherosclerotic cardiovascular diseases. However, translating the findings from animal models to humans has inherent limitations, necessitating a novel platform. Therefore, herein, an arterial model is established using a microphysiological system. This model successfully replicates the stratified characteristics of human arteries by integrating collagen, endothelial cells (ECs), and vascular smooth muscle cells (VSMCs). Perfusion via a peristaltic pump shows dynamic characteristics distinct from those of static culture models. High glucose, advanced glycation end products (AGEs), and interleukin-1 beta are employed to stimulate diabetic conditions, resulting in notable cellular changes and different levels of cytokines and nitric oxide. Additionally, the interactions between the disease models and oxidized low-density lipoproteins (LDL) are examined. Finally, the potential therapeutic effects of metformin, atorvastatin, and diphenyleneiodonium are investigated. Metformin and diphenyleneiodonium mitigate high-glucose- and AGE-associated pathological changes, whereas atorvastatin affects only the morphology of ECs. Altogether, the arterial model represents a pivotal advancement, offering a robust and insightful platform for investigating cardiovascular diseases and their corresponding drug development.

The development of diphenyleneiodonium analogs as GPR3 agonists.

G protein-coupled receptor 3 (GPR3) is an orphan receptor potentially involved in many important physiological processes such as drug abuse, neuropathic pain, and anxiety and depression related disorders. Pharmacological studies of GPR3 have been limited due to the restricted number of known agonists and inverse agonists for this constitutively active receptor. In this medicinal chemistry study, we report the discovery of GPR3 agonists based off the diphenyleneiodonium (DPI) scaffold. The most potent full agonist was the 3-trifluoromethoxy analog (32) with an EC50 of 260 nM and 90% efficacy compared to DPI. Investigation of a homology model of GPR3 from multiple sequence alignment resulted in the finding of a binding site rich in potential π-π and π-cation interactions stabilizing DPI-scaffold agonists. MMGBSA free energy analysis showed a good correlation with trends in observed EC50s. DPI analogs retained the same high receptor selectivity for GPR3 over GPR6 and GPR12 as observed with DPI. Collectively, the DPI analog series shows that order of magnitude improvements in potency with the scaffold were attainable; however, attempts to replace the iodonium ion to make the scaffold more druggable failed.

Reaction of Diaryliodonium Salts with Potassium Alkyl Xanthates as an Entry Point to Accessing Organosulfur Compounds.

Preparation of S-aryl xanthates via transition-metal-catalyzed or SNAr reactions is complicated by their further transformations under the utilized conditions. In contrast, S-arylation of potassium O-alkyl xanthates with diaryliodonium salts proceeds under mild conditions, enabling access to substituted S-aryl xanthates. The method exhibits good functional group tolerance and can be applied to the late-stage C-H functionalization of drug molecules. Divergent transformations of the resulting S-aryl xanthates provide rapid access to a range of medicinal chemistry-relevant organosulfur compounds.

Tetraphenylphosphonium Chloride-Enhanced Ionization Coupled to Orbitrap Mass Spectrometry for Sensitive and Non-targeted Screening of Polyhalogenated Alkyl Compounds from Limited Serum.

Although many types of halogenated compounds are known to bioaccumulate in humans, few are routinely biomonitored and many have remained uncharacterized in human exposome studies due to a lack of high-sensitivity and high-resolution analytical methods. In this study, we discovered tetraphenylphosphonium chloride (Ph4PCl, 10 µM) as a simple additive to the mobile phase, which enhanced the ionizations of polyhalogenated alkyl compounds (such as organochlorinated pesticides [OCPs], chlorinated paraffins [CPs], dechlorane plus [DPs], and some brominated flame retardants [BFRs]) in the form [M + Cl]- and boosted mass spectrometry responses by an average of 1-3 orders of magnitude at a resolution of 140,000. Ph4PCl-enhanced ionization coupled with a halogenation-guided screening process was used to establish a sensitive and non-targeted method that required only single-step sample preparation and identified Cl- and/or bromine-containing alkyl compounds. The method enabled the identification of ∼700 polyhalogenated compounds from 200 µL of human serum, 240 of which were known compounds: 33 short-chain CPs, 52 median-chain CPs, 97 long-chain CPs, 22 very short-chain CPs (vSCCPs), 19 OCPs, 13 DPs, and 4 BFRs. We also identified 325 emerging contaminants (34 unsaturated CPs, 285 chlorinated fatty acid methyl esters [CFAMEs], and 6 chloro-bromo alkenes) and 130 new contaminants (114 oxygen-containing CPs, 2 hexachlorocyclohexane structural analogs, and 11 amino-containing and 3 nitrate-containing chlorinated compounds). The full scan results highlighted the dominance of CPs, CFAMEs, vSCCPs, and dichlorodiphenyltrichloroethanes in the serum samples. Ph4PCl-enhanced ionization enabled the sensitive and non-targeted identifications of polyhalogenated compounds in small volumes of biological fluid.

Diphenyleneiodonium Treatment Inhibits the Development of Severe Herpes Stromal Keratitis Lesions.

Reactive oxygen species (ROS) play an important role in tissue inflammation. In this study, we measured the intracellular level of ROS in herpes stromal keratitis (HSK) corneas and determined the outcome of manipulating ROS level on HSK severity. Our results showed the predominance of ROS generation in neutrophils but not CD4 T cells in HSK corneas. NADPH oxidase 2 (NOX2) enzyme is known to generate ROS in myeloid cells. Our results showed baseline expression of different NOX2 subunits in uninfected corneas. After corneal herpes simplex virus-1 (HSV-1) infection, an enhanced expression of NOX2 subunits was detected in infected corneas. Furthermore, flow cytometry results showed a higher level of gp91 (Nox2 subunit) protein in neutrophils from HSK corneas, suggesting the involvement of NOX2 in generating ROS. However, no significant decrease in ROS level was noticed in neutrophils from HSV-1-infected gp91-/- mice than in C57BL/6J (B6) mice, suggesting NOX2 is not the major contributor in generating ROS in neutrophils. Next, we used diphenyleneiodonium (DPI), a flavoenzyme inhibitor, to pharmacologically manipulate the ROS levels in HSV-1-infected mice. Surprisingly, the neutrophils from peripheral blood and corneas of the DPI-treated group exhibited an increased level of ROS than the vehicle-treated group of infected B6 mice. Excessive ROS is known to cause cell death. Accordingly, DPI treatment resulted in a significant decrease in neutrophil frequency in peripheral blood and corneas of infected mice and was associated with reduced corneal pathology. Together, our results suggest that regulating ROS levels in neutrophils can ameliorate HSK severity. IMPORTANCE Neutrophils are one of the primary immune cell types involved in causing tissue damage after corneal HSV-1 infection. This study demonstrates that intracellular ROS production in the neutrophils in HSK lesions is not NOX2 dependent. Furthermore, manipulating ROS levels in neutrophils ameliorates the severity of HSK lesions. Our findings suggest that excessive intracellular ROS in neutrophils disrupt redox homeostasis and affect their survival, resulting in a decrease in HSK lesion severity.

Metal-Free Regioselective N2-Arylation of 1H-Tetrazoles with Diaryliodonium Salts.

We describe a simple, metal-free regioselective N2-arylation strategy for 5-substituted-1H-tetrazoles with diaryliodonium salts to access 2-aryl-5-substituted-tetrazoles. Diaryliodonium salts with a wide range of both electron-rich and previously challenged electron-deficient aryl groups are applicable in this method. Diversely functionalized tetrazoles are tolerable also. We have devised a one-pot system to synthesize 2,5-diaryl-tetrazoles directly from nitriles. The synthetic utility of this method is furthered extended to late-stage arylation of two biologically active molecules.

Toxicity studies of select ionic liquids (1-ethyl-3-methylimidazolium chloride, 1-butyl-3-methylimidazolium chloride, 1-butyl-1-methylpyrrolidinium chloride, and n-butylpyridinium chloride) administered in drinking water to Sprague Dawley (Hsd:Sprague Dawley SD) rats and B6C3F1/N mice.

Ionic liquids (ILs) are synthetic solvents with applications in a variety of industrial and chemical industries. Human exposure to this diverse chemical class is primarily through dermal or oral routes. Research suggests toxicity may be associated with IL structural characteristics, including the type of cation base or alkyl chain substitutions associated with the cation. To further investigate this hypothesis, the National Toxicology Program (NTP) conducted 3-month toxicity studies in male and female Sprague Dawley (Hsd:Sprague Dawley SD) rats and B6C3F1/N mice (n = 10/sex/exposure group; 3 exposure concentrations per IL) to compare the relative toxicities of four ILs administered via drinking water-1-ethyl-3-methylimidazolium chloride (Emim-Cl), 1-butyl-3-methylimidazolium chloride (Bmim-Cl), 1-butyl-1-methylpyrrolidinium chloride (Bmpy-Cl), and n-butylpyridinium chloride (NBuPy-Cl). (Abstract Abridged).

Metal-Free C-H Functionalization via Diaryliodonium Salts with a Chemically Robust Dummy Ligand.

A two-step strategy for the transition-metal-free C-H functionalization of arenes using unsymmetrical iodonium salts as versatile synthetic linchpins is presented. The key to the success of this strategy is the identification of the 3,5-dimethyl-4-isoxazolyl (DMIX) group as a superior dummy ligand, which enables not only site-selective C-H functionalization to afford unsymmetrical iodonium salts, but also highly selective aryl transfer during the subsequent metal-free coupling reaction. Both electron-rich and moderately electron-deficient arenes can be converted into the iodonium salts through C-H functionalization, allowing for diverse structural elaboration by metal-free C-N, C-C, C-S, and C-O coupling.

Metal-free S-arylation of 5-mercaptotetrazoles and 2-mercaptopyridine with unsymmetrical diaryliodonium salts.

Herein, we demonstrate the application of unsymmetrical iodonium salts towards S-arylation of heterocyclic thiols (especially tetrazole-5-thiols and pyridine-2-thiol) under metal-free conditions, affording a diverse range of di(hetero)aryl thioethers in moderate to good yields. A detailed study on the effects of counter-anions and the auxiliary of iodonium salts was conducted. Suitable auxiliary selection of the unsymmetrical iodonium salt offers flexibility for a wide range of aryl moieties and its incorporation into S-arylation. The DFT study supports the experimental observations of chemoselective arylation.

Combined Levo-tetrahydropalmatine and diphenyleneiodonium chloride enhances antitumor activity in hepatocellular carcinoma.

Metabolic dysregulation is a hallmark of hepatocellular carcinoma (HCC). AMPK is a crucial hub of metabolic regulation during cancer progression. We show that phytochemical Levo-tetrahydropalmatine (THP) activates AMPK-dependent autophagy to downregulate the mitochondrial respiration and glycolysis. Consequently, THP significantly decreased cell viability in two HCC cell lines, BEL-7402 and SMMC-7721. Similarly, NOX4 inhibitor diphenyleneiodonium chloride (DPI) induces concomitant downregulation of the mitochondrial and glycolytic metabolism. In contrast to THP, cells are less sensitive to proliferation inhibition induced by DPI treatment as compared to THP treatment did. Combined treatment of THP and DPI was found to be more efficacious in killing cancer cells than either of the agents treated individually. Indeed, the co-operative effect by the THP-DPI combination improves the pro-apoptotic activity in response to the energy depletion as outlined by a drastic decrease in ATP levels. Therapeutic regime significantly reduced the tumor growth in mice. Importantly, this is realized without causing systemic toxicity to other organs. Collectively, our work shows that the combinatorial therapy of autophagy activator THP and NOX4 inhibitor DPI may be considered as a therapeutic avenue against HCC.

One-Pot Synthesis of Heteroatom-Bridged Cyclic Diaryliodonium Salts.

Two one-pot procedures for the construction of O- and N-bridged diaryliodonium triflates are described. An effective aryne-mediated arylation of o-iodophenols and -sulfonamides provides diarylether and diarylamine intermediates, which are subsequently oxidized and cyclized to the corresponding diaryliodaoxinium and -iodazinium salts. Different derivatizations were applied to demonstrate their capacity as useful building blocks and gain a deeper understanding toward the general reactivity of these underdeveloped but potentially highly useful compounds.

Diphenyleneiodonium efficiently inhibits the characteristics of a cancer stem cell model derived from induced pluripotent stem cells.

Diphenyleneiodonium (DPI) has long been evaluated as an anticancer drug inhibiting NADPH oxidase, the IC50 in several cancer cell lines was reported 10 µM, which is too high for efficacy. In this study, we employed miPS-Huh7cmP cells, which we previously established as a cancer stem cell (CSC) model from induced pluripotent stem cells, to reevaluate the efficacy of DPI because CSCs are currently one of the main foci of therapeutic strategy to treat cancer, but generally considered resistant to chemotherapy. As a result, the conventional assay for the cell growth inhibition by DPI accounted for an IC50 at 712 nM that was not enough to define the effectiveness as an anticancer drug. Simultaneously, the wound-healing assay revealed an IC50 of approximately 500 nM. Comparatively, the IC50 values shown on sphere formation, colony formation, and tube formation assays were 5.52, 12, and 8.7 nM, respectively. However, these inhibitory effects were not observed by VAS2780, also a reputed NADPH oxidase inhibitor. It is noteworthy that these three assays are evaluating the characteristic of CSCs and are designed in the three-dimensional (3D) culture methods. We concluded that DPI could be a suitable candidate to target mitochondrial respiration in CSCs. We propose that the 3D culture assays are more efficient to screen anti-CSC drug candidates and better mimic tumor microenvironment when compared to the adherent monolayer of 2D culture system used for a conventional assay, such as cell growth inhibition and wound-healing assays.

High-pH structure of EmrE reveals the mechanism of proton-coupled substrate transport.

The homo-dimeric bacterial membrane protein EmrE effluxes polyaromatic cationic substrates in a proton-coupled manner to cause multidrug resistance. We recently determined the structure of substrate-bound EmrE in phospholipid bilayers by measuring hundreds of protein-ligand HN-F distances for a fluorinated substrate, 4-fluoro-tetraphenylphosphonium (F4-TPP+), using solid-state NMR. This structure was solved at low pH where one of the two proton-binding Glu14 residues is protonated. Here, to understand how substrate transport depends on pH, we determine the structure of the EmrE-TPP complex at high pH, where both Glu14 residues are deprotonated. The high-pH complex exhibits an elongated and hydrated binding pocket in which the substrate is similarly exposed to the two sides of the membrane. In contrast, the low-pH complex asymmetrically exposes the substrate to one side of the membrane. These pH-dependent EmrE conformations provide detailed insights into the alternating-access model, and suggest that the high-pH conformation may facilitate proton binding in the presence of the substrate, thus accelerating the conformational change of EmrE to export the substrate.

Membrane Permeability of Modified Butyltriphenylphosphonium Cations.

The alkyltriphenylphosphonium (TPP) group is the most widely used vector targeted to mitochondria. Previously, the length of the alkyl linker was varied as well as structural modifications in the TPP phenyl rings to obtain the optimal therapeutic effect of a pharmacophore conjugated with a lipophilic cation. In the present work, we synthesized butyltriphenylphosphonium cations halogenated and methylated in phenyl rings (C4TPP-X) and measured electrical current through a planar lipid bilayer in the presence of C4TPP-X. The permeability of C4TPP-X varied in the range of 6 orders of magnitude and correlates well with the previously measured translocation rate constant for dodecyltriphenylphosphonium analogues. The partition coefficient of the butyltriphenylphosphonium analogues obtained by calculating the difference in the free energy of cation solvation in water and octane using quantum chemical methods correlates well with the permeability values. Using an ion-selective electrode, a lower degree of accumulation of analogues with halogenated phenyl groups was found on isolated mitochondria of rat liver, which is in agreement with their permeability decrease. Our results indicate the translocation of the butyltriphenylphosphonium cations across the hydrophobic membrane core as rate-limiting stage in the permeability process rather than their binding/release to/from the membrane.

Influence of cellular redox environment on aryl hydrocarbon receptor ligands induced melanogenesis.

Many environmental pollutants, natural compounds, as well as endogenous chemicals exert their biological/toxicological effects by reacting with the aryl hydrocarbon receptor (AhR). Previous evidence shed new light on the role of AhR in skin physiology by regulating melanin production. In this study, we investigated the effect of oxidative imbalance induced by AhR ligands on the melanogenesis process in B16 murine melanoma cells. Exposure to 6-formylindolo[3,2-b] carbazole (FICZ) or benzo-α-pyrene (BαP) led to enhanced expression of CTNNB1, MITF, and TYR genes following increased tyrosinase enzyme activity and melanin content in an AhR-dependent manner. Analysis of the presence of reactive oxygen species (ROS) as well as reduced glutathione (GSH) / oxidized glutathione (GSSG) ratio revealed that treatment with AhR ligands is associated with oxidative stress which can be ameliorated with NAC (N-acetyl cysteine) or diphenyleneiodonium chloride (DPI). On the other hand, NAC and DPI enhanced melanogenesis induced by AhR ligands by reducing the level of ROS. We have shown for the first time that a cellular redox status is a critical event during AhR ligand-induced melanogenesis.

Photocatalytic cross-couplings via the cleavage of N-O bonds.

N-(Acyloxy)phthalimide and oxime derivatives containing N-O bonds are important chemicals and synthetic intermediates, and visible light photoredox reductions of the N-O bonds provide carbon- or nitrogen-centered radicals for N-(acyloxy)phthalimide derivatives and iminyl radicals for oxime derivatives. This feature article summarises the recent progress in the visible light photoredox organic reactions, including decarboxylative addition reactions, alkylation, allylation, alkenylation, alkynylation, arylation, heteroarylation and cascade annulation of N-(acyloxy)phthalimide derivatives through the formation of carbon-carbon bonds, decarboxylative borylation, amination, oxygenation, sulfuration, selenylation, fluorination and iodination of N-(acyloxy)phthalimide derivatives through the formation of carbon-heteroatom bonds, and additions to arenes and alkenes, hydrogen atom transfer and the cleavage of α-carbon-carbon bonds via the iminyl radical intermediates for oxime derivatives.

Inhibition of phase-1 biotransformation and cytostatic effects of diphenyleneiodonium on hepatoblastoma cell line HepG2 and a CYP3A4-overexpressing HepG2 cell clone.

Cell-based in vitro liver models are an important tool in the development and evaluation of new drugs in pharmacological and toxicological drug assessment. Hepatic microsomal enzyme complexes, consisting of cytochrome P450 oxidoreductase (CPR) and cytochrome P450 monooxygenases (CYPs), play a decisive role in catalysing phase-1 biotransformation of pharmaceuticals and xenobiotics. For a comprehensive understanding of the phase-1 biotransformation of drugs, the availability of well-characterized substances for the targeted modulation of in vitro liver models is essential. In this study, we investigated diphenyleneiodonium (DPI) for its ability to inhibit phase-1 enzyme activity and further its toxicological profile in an in vitro HepG2 cell model with and without recombinant expression of the most important drug metabolization enzyme CYP3A4.Aim of the study was to identify effective DPI concentrations for CPR/CYP activity modulation and potentially associated dose and time dependent hepatotoxic effects. The cells were treated with DPI doses up to 5,000nM (versus vehicle control) for a maximum of 48 h and subsequently examined for CYP3A4 activity as well as various toxicological relevant parameters such as cell morphology, integrity and viability, intracellular ATP level, and proliferation. Concluding, the experiments revealed a time- and concentration-dependent DPI mediated partial and complete inhibition of CYP3A4 activity in CYP3A4 overexpressing HepG2-cells (HepG2-CYP3A4). Other cell functions, including ATP synthesis and consequently the proliferation were negatively affected in both in vitro cell models. Since neither cell integrity nor cell viability were reduced, the effect of DPI in HepG2 can be assessed as cytostatic rather than cytotoxic.