Development of Membrane-Targeting Fluorescent 2-Phenyl-1H-phenanthro[9,10-d]imidazole-Antimicrobial Peptide Mimic Conjugates against Methicillin-Resistant Staphylococcus aureus.

The escalation of multidrug-resistant bacterial infections, especially infections caused by methicillin-resistant Staphylococcus aureus (MRSA), underscores the urgent need for novel antimicrobial drugs. Here, we synthesized a series of amphiphilic 2-phenyl-1H-phenanthro[9,10-d]imidazole-antimicrobial peptide (AMP) mimic conjugates (III1-30). Among them, compound III13 exhibited excellent antibacterial activity against G+ bacteria and clinical MRSA isolates (MIC = 0.5-2 µg/mL), high membrane selectivity, and low toxicity. Additionally, compared with traditional clinical antibiotics, III13 demonstrated rapid bactericidal efficacy and was less susceptible to causing bacterial resistance. Mechanistic studies revealed that III13 targets phosphatidylglycerol (PG) on bacterial membranes to disrupt membrane integrity, leading to an increase in intracellular ROS and leakage of proteins and DNA, ultimately causing bacterial cell death. Furthermore, III13 possessed good fluorescence properties with potential for further dynamic monitoring of the antimicrobial process. Notably, III13 showed better in vivo efficacy against MRSA compared to vancomycin, suggesting its potential as a promising candidate for anti-MRSA medication.

LSD1-Based Reversible Inhibitors Virtual Screening and Binding Mechanism Computational Study.

As one of the crucial targets of epigenetics, histone lysine-specific demethylase 1 (LSD1) is significant in the occurrence and development of various tumors. Although several irreversible covalent LSD1 inhibitors have entered clinical trials, the large size and polarity of the FAD-binding pocket and undesired toxicity have focused interest on developing reversible LSD1 inhibitors. In this study, targeting the substrate-binding pocket of LSD1, structure-based and ligand-based virtual screenings were adopted to expand the potential novel structures with molecular docking and pharmacophore model strategies, respectively. Through drug-likeness evaluation, ADMET screening, molecular dynamics simulations, and binding free energy screening, we screened out one and four hit compounds from the databases of 2,029,554 compounds, respectively. Generally, these hit compounds can be divided into two categories, amide (Lig2 and Comp2) and 1,2,4-triazolo-4,3-α-quinazoline (Comp3, Comp4, Comp7). Among them, Comp4 exhibits the strongest binding affinity. Finally, the binding mechanisms of the hit compounds were further calculated in detail by the residue free energy decomposition. It was found that van der Waals interactions contribute most to the binding, and FAD is also helpful in stabilizing the binding and avoiding off-target effects. We believe this work not only provides a solid theoretical foundation for the design of LSD1 substrate reversible inhibitors, but also expands the diversity of parent nucleus, offering new insights for synthetic chemists.

New insights into the proton pumping mechanism of ba3 cytochrome c oxidase: the functions of key residues and water.

As the terminal oxidase of cell respiration in mitochondria and aerobic bacteria, the proton pumping mechanism of ba3-type cytochrome c oxidase (CcO) of Thermus thermophiles is still not fully understood. Especially, the functions of key residues which were considered as the possible proton loading sites (PLSs) above the catalytic center, as well as water located above and within the catalytic center, remain unclear. In this work, molecular dynamic simulations were performed on a set of designed mutants of key residues (Asp287, Asp372, His376, and Glu126II). The results showed that Asp287 may not be a PLS, but it could modulate the ability of the proton transfer pathway to transfer protons through its salt bridge with Arg225. Maintaining the closed state of the water pool above the catalytic center is necessary for the participation of inside water molecules in proton transfer. Water molecules inside the water pool can form hydrogen bond chains with PLS to facilitate proton transfer. Additional quantum cluster models of the Fe-Cu metal catalytic center are established, indicating that when the proton is transferred from Tyr237, it is more likely to reach the OCu atom directly through only one water molecule. This work provides a more profound understanding of the functions of important residues and specific water molecules in the proton pumping mechanism of CcO.

Phenothiazine-Based LSD1 Inhibitor Promotes T-Cell Killing Response of Gastric Cancer Cells.

Histone lysine specific demethylase 1 (LSD1) has been recognized as an important epigenetic target for cancer treatment. Although several LSD1 inhibitors have entered clinical trials, the discovery of novel potent LSD1 inhibitors remains a challenge. In this study, the antipsychotic drug chlorpromazine was characterized as an LSD1 inhibitor (IC50 = 5.135 µM), and a series of chlorpromazine derivatives were synthesized. Among them, compound 3s (IC50 = 0.247 µM) was the most potent one. More importantly, compound 3s inhibited LSD1 in the cellular level and downregulated the expression of programmed cell death-ligand 1 (PD-L1) in BGC-823 and MFC cells to enhance T-cell killing response. An in vivo study confirmed that compound 3s can inhibit MFC cell proliferation without significant toxicity in immunocompetent mice. Taken together, our findings indicated that the novel LSD1 inhibitor 3s tethering a phenothiazine scaffold may serve as a lead compound for further development to activate T-cell immunity in gastric cancer.

Targeting Tumor Necrosis Factor Receptor 1 with Selected Aptamers for Anti-Inflammatory Activity.

Tumor necrosis factor-α (TNFα) inhibitors are widely used in treating autoimmune diseases like rheumatoid arthritis (RA). These inhibitors can presumably alleviate RA symptoms by blocking TNFα-TNF receptor 1 (TNFR1)-mediated pro-inflammatory signaling pathways. However, the strategy also interrupts the survival and reproduction functions conducted by TNFα-TNFR2 interaction and causes side effects. Thus, it is urgently needed to develop inhibitors that can selectively block TNFα-TNFR1 but not TNFα-TNFR2. Here, nucleic acid-based aptamers against TNFR1 are explored as potential anti-RA candidates. Through the systematic evolution of ligands by exponential enrichment (SELEX), two types of TNFR1-targeting aptamers were obtained, and their KD values are approximately 100-300 nM. In silico analysis shows that the binding interface of aptamer-TNFR1 highly overlapped with natural TNFα-TNFR1 binding. On the cellular level, the aptamers can exert TNFα inhibitory activity by binding to TNFR1. The anti-inflammatory efficiencies of aptamers were assessed and further enhanced using divalent aptamer constructs. These findings provide a new strategy to block TNFR1 for potential anti-RA treatment precisely.

Protein DEK and DTA Aptamers: Insight Into the Interaction Mechanisms and the Computational Aptamer Design.

By blocking the DEK protein, DEK-targeted aptamers (DTAs) can reduce the formation of neutrophil extracellular traps (NETs) to reveal a strong anti-inflammatory efficacy in rheumatoid arthritis. However, the poor stability of DTA has greatly limited its clinical application. Thus, in order to design an aptamer with better stability, DTA was modified by methoxy groups (DTA_OMe) and then the exact DEK-DTA interaction mechanisms were explored through theoretical calculations. The corresponding 2'-OCH3-modified nucleotide force field was established and the molecular dynamics (MD) simulations were performed. It was proved that the 2'-OCH3-modification could definitely enhance the stability of DTA on the premise of comparative affinity. Furthermore, the electrostatic interaction contributed the most to the binding of DEK-DTA, which was the primary interaction to maintain stability, in addition to the non-specific interactions between positively-charged residues (e.g., Lys and Arg) of DEK and the negatively-charged phosphate backbone of aptamers. The H-bond network analysis reminded that eight bases could be mutated to probably enhance the affinity of DTA_OMe. Therein, replacing the 29th base from cytosine to thymine of DTA_OMe was theoretically confirmed to be with the best affinity and even better stability. These research studies imply to be a promising new aptamer design strategy for the treatment of inflammatory arthritis.

Characterization of erm(B)-carrying Campylobacter Spp. of retail chicken meat origin.

OBJECTIVES: The erm(B) gene in Campylobacter Spp., conferring resistance to macrolides, is of great concern worldwide. In this study, the prevalence of erm(B) in Campylobacter of retail chicken meat origin was investigated and the characterization of erm(B)-harboring Campylobacter isolates was analyzed. METHODS: Antimicrobial susceptibility testing was performed to determine the susceptibility of Campylobacter isolates. Whole-genome sequencing and analysis were used to characterize sequence type (ST) and genetic context of erm(B). Natural transformation was conducted to evaluate transferability of the erm(B) gene. RESULTS: A total of 16 (11.8%) Campylobacter isolates were obtained from 136 samples collected from retail chicken meat, amongst which five erm(B)-positive isolates were identified as Campylobacter coli belonging to ST3753 (n = 4) and ST825 (n = 1). A total of 22 Campylobacter Spp. were erm(B)-positive in GenBank database; all isolates were collected in China except for one Campylobacter jejuni isolate. Diverse STs were involved in these erm(B)-carrying isolates. Comparison analysis indicated that 11 types of genetic environment for erm(B) were identified, mostly associated with multidrug-resistance genomic islands (MDRGIs). The genetic context of erm(B) in C. coli of retail chicken meat origin showed high nucleotide sequence similarity with that of C. coli from humans. CONCLUSION: This is the first report of prevalence and characterization for erm(B) in Campylobacter of retail chicken meat origin. The genetic context of erm(B) in C. coli isolates from retail chicken meat is highly homologous with that of C. coli from humans; this impies the possibility of zoonotic transmission of erm(B) in Campylobacter, which presents a threat to public health.

Discovery of cinnamamide-barbiturate hybrids as a novel class of Nrf2 activator against myocardial ischemia/reperfusion injury.

Myocardial ischemia/reperfusion (MI/R) has been a challenge for global public health. Activation of nuclear factor erythroid-2-related factor 2 (Nrf2) signaling could attenuate MI/R injury by maintaining cell redox balance and reducing oxidative damage. Cinnamamide derivatives have been proven to be a class of potential Nrf2 activators and cardioprotective agents. The development of novel cinnamamide derivatives to combat oxidative stress in cardiomyocytes is highly desirable. In this study, twenty-three cinnamamide-barbiturate hybrids were studied. Cell-based assays showed that most of the compounds exhibited excellent protective activity against H2O2-induced oxidative injury in H9c2 cells. Notably, compound 7w, which had the highest activity and low cytotoxicity, was demonstrated to remarkably reduce intracellular ROS accumulation by activating the mRNA expression of Nrf2 and its downstream antioxidant gene HO-1, indicating a novel promising antioxidant and Nrf2 activator. The probable binding mode between protein Keap1 and compound 7w was also studied via molecule docking. Furthermore, we found that the administration of compound 7w could significantly reduce the cardiac infarct size and improve the cardiac function against MI/R injury in rats, as well as decrease cardiac oxidative stress. Taken together, we report, for the first time, that cinnamamide-barbiturate hybrids are a novel class of potential cardioprotective agents. The excellent cardioprotective action of such compounds rely on enhancing the endogenous antioxidative system by upregulating the Nrf2 signaling pathway in vitro and in vivo against MI/R damage. These findings provide a new perspective for designing cinnamamide-barbiturate hybrids as a novel class of Nrf2 activator against cardiovascular diseases.

Angiogenesis and anti-leukaemia activity of novel indole derivatives as potent colchicine binding site inhibitors.

The screened compound DYT-1 from our in-house library was taken as a lead (inhibiting tubulin polymerisation: IC50=25.6 µM, anti-angiogenesis in Zebrafish: IC50=38.4 µM, anti-proliferation against K562 and Jurkat: IC50=6.2 and 7.9 µM, respectively). Further investigation of medicinal chemistry conditions yielded compound 29e (inhibiting tubulin polymerisation: IC50=4.8 µM and anti-angiogenesis in Zebrafish: IC50=3.6 µM) based on tubulin and zebrafish assays, which displayed noteworthily nanomolar potency against a variety of leukaemia cell lines (IC50= 0.09-1.22 µM), especially K562 cells where apoptosis was induced. Molecular docking, molecular dynamics (MD) simulation, radioligand binding assay and cellular microtubule networks disruption results showed that 29e stably binds to the tubulin colchicine site. 29e significantly inhibited HUVEC tube formation, migration and invasion in vitro. Anti-angiogenesis in vivo was confirmed by zebrafish xenograft. 29e also prominently blocked K562 cell proliferation and metastasis in blood vessels and surrounding tissues of the zebrafish xenograft model. Together with promising physicochemical property and metabolic stability, 29e could be considered an effective anti-angiogenesis and -leukaemia drug candidate that binds to the tubulin colchicine site.

Cardioprotective effect of MLN4924 on ameliorating autophagic flux impairment in myocardial ischemia-reperfusion injury by Sirt1.

Neddylation is essential for cardiomyocyte survival in the presence of oxidative stress, and it participates in autophagy regulation. However, whether MLN4924-an inhibitor of neddylation-exerts cardioprotective effects against myocardial ischemia/reperfusion (MI/R) remains unknown. In the present study, MLN4924 exerted strong cardioprotective effects, demonstrated by significantly elevated cell viability, a decreased LDH leakage rate, and improved cell morphology following H2O2-induced injury in vitro. MLN4924 also markedly decreased the serum myocardial zymogram level, ameliorated cardiac histopathological alterations, and alleviated left ventricular contractile dysfunction, thus limiting the cardiac infarct size in vivo compared with those in MI/R mice. Amazingly, such action of MLN4924 was abrogated by a combined treatment with the autophagic flux inhibitor, chloroquine. The mRFP-GFP-LC3 assay illustrated that MLN4924 restored the defective autophagic flux via enhancing the autolysosome formation. Notably, the expression levels of Rab7 and Atg5 were markedly up-regulated in MLN4924 treated cells and mice subjected to H2O2 or MI/R, respectively, while knockdown of Sirt1 in cells and heart tissue largely blocked such effect and induced autophagosome accumulation by inhibiting its fusion with lysosomes. Transmission electron microscopic analysis, histopathological assay and TUNEL detection of the heart tissues showed that the absence of Sirt1 blocked the cardioprotective effect of MLN4924 by further exacerbating the impaired autophagic flux during MI/R injury in vivo. Taken together, MLN4924 exhibited the strong cardioprotective action via restoring the impaired autophagic flux in H2O2-induced injury in vitro and in MI/R mice. Our work implicated that Sirt1 played a critical role in autophagosome clearance, likely through up-regulating Rab7 in MI/R.

Data for molecular dynamics simulations of B-type cytochrome c oxidase with the Amber force field.

Cytochrome c oxidase (CcO) is a vital enzyme that catalyzes the reduction of molecular oxygen to water and pumps protons across mitochondrial and bacterial membranes. This article presents parameters for the cofactors of ba3-type CcO that are compatible with the all-atom Amber ff12SB and ff14SB force fields. Specifically, parameters were developed for the CuA pair, heme b, and the dinuclear center that consists of heme a3 and CuB bridged by a hydroperoxo group. The data includes geometries in XYZ coordinate format for cluster models that were employed to compute proton transfer energies and derive bond parameters and point charges for the force field using density functional theory. Also included are the final parameter files that can be employed with the Amber leap program to generate input files for molecular dynamics simulations with the Amber software package. Based on the high resolution (1.8 Å) X-ray crystal structure of the ba3-type CcO from Thermus thermophilus (Protein Data Bank ID number PDB: 3S8F), we built a model that is embedded in a POPC lipid bilayer membrane and solvated with TIP3P water molecules and counterions. We provide PDB data files of the initial model and the equilibrated model that can be used for further studies.

Water exit pathways and proton pumping mechanism in B-type cytochrome c oxidase from molecular dynamics simulations.

Cytochrome c oxidase (CcO) is a vital enzyme that catalyzes the reduction of molecular oxygen to water and pumps protons across mitochondrial and bacterial membranes. While proton uptake channels as well as water exit channels have been identified for A-type CcOs, the means by which water and protons exit B-type CcOs remain unclear. In this work, we investigate potential mechanisms for proton transport above the dinuclear center (DNC) in ba3-type CcO of Thermus thermophilus. Using long-time scale, all-atom molecular dynamics (MD) simulations for several relevant protonation states, we identify a potential mechanism for proton transport that involves propionate A of the active site heme a3 and residues Asp372, His376 and Glu126(II), with residue His376 acting as the proton-loading site. The proposed proton transport process involves a rotation of residue His376 and is in line with experimental findings. We also demonstrate how the strength of the salt bridge between residues Arg225 and Asp287 depends on the protonation state and that this salt bridge is unlikely to act as a simple electrostatic gate that prevents proton backflow. We identify two water exit pathways that connect the water pool above the DNC to the outer P-side of the membrane, which can potentially also act as proton exit transport pathways. Importantly, these water exit pathways can be blocked by narrowing the entrance channel between residues Gln151(II) and Arg449/Arg450 or by obstructing the entrance through a conformational change of residue Tyr136, respectively, both of which seem to be affected by protonation of residue His376.

A broken-symmetry density functional study of structures, energies, and protonation states along the catalytic O-O bond cleavage pathway in ba3 cytochrome c oxidase from Thermus thermophilus.

Broken-symmetry density functional calculations have been performed on the [Fea3, CuB] dinuclear center (DNC) of ba3 cytochrome c oxidase from Thermus thermophilus in the states of [Fea3(3+)-(HO2)(-)-CuB(2+), Tyr237(-)] and [Fea3(4+)[double bond, length as m-dash]O(2-), OH(-)-CuB(2+), Tyr237˙], using both PW91-D3 and OLYP-D3 functionals. Tyr237 is a special tyrosine cross-linked to His233, a ligand of CuB. The calculations have shown that the DNC in these states strongly favors the protonation of His376, which is above propionate-A, but not of the carboxylate group of propionate-A. The energies of the structures obtained by constrained geometry optimizations along the O-O bond cleavage pathway between [Fea3(3+)-(O-OH)(-)-CuB(2+), Tyr237(-)] and [Fea3(4+)[double bond, length as m-dash]O(2-)HO(-)-CuB(2+), Tyr237˙] have also been calculated. The transition of [Fea3(3+)-(O-OH)(-)-CuB(2+), Tyr237(-)] → [Fea3(4+)[double bond, length as m-dash]O(2-)HO(-)-CuB(2+), Tyr237˙] shows a very small barrier, which is less than 3.0/2.0 kcal mol(-1) in PW91-D3/OLYP-D3 calculations. The protonation state of His376 does not affect this O-O cleavage barrier. The rate limiting step of the transition from state A (in which O2 binds to Fea3(2+)) to state PM ([Fea3(4+)[double bond, length as m-dash]O(2-), OH(-)-CuB(2+), Tyr237˙], where the O-O bond is cleaved) in the catalytic cycle is, therefore, the proton transfer originating from Tyr237 to O-O to form the hydroperoxo [Fea3(3+)-(O-OH)(-)-CuB(2+), Tyr237(-)] state. The importance of His376 in proton uptake and the function of propionate-A/neutral-Asp372 as a gate to prevent the proton from back-flowing to the DNC are also shown.

Recent advances in carbon dioxide capture, fixation, and activation by using N-heterocyclic carbenes.

In the last two decades, CO2 emission has caused a lot of environmental problems. To mitigate the concentration of CO2 in the atmosphere, various strategies have been implemented, one of which is the use of N-heterocyclic carbenes (NHCs) and related complexes to accomplish the capture, fixation, and activation of CO2 effectively. In this review, we summarize CO2 capture, fixation, and activation by utilizing NHCs and related complexes; homogeneous reactions and their reaction mechanisms are discussed. Free NHCs and NHC salts can capture CO2 in both direct and indirect ways to form imidazolium carboxylates, and they can also catalyze the reaction of aromatic aldehydes with CO2 to form carboxylic acids and derivatives. Moreover, associated with transition metals (TMs), NHCs can form NHC-TM complexes to transform CO2 into industrial acid or esters. Non-metal-NHC complexes can also catalyze the reactions of silicon and boron complexes with CO2 . In addition, catalytic cycloaddition of epoxides with CO2 is another effective function of NHC complexes, and NHC ionic liquids perform excellently in this aspect.

The reduction of carbon dioxide in iron biocatalyst catalytic hydrogenation reaction: a theoretical study.

The reaction mechanism of CO2 hydrogenation catalyzed by [FeH(PP3)]BF4 (PP3 = P(CH2CH2PPh2)3) had been investigated by DFT calculations. Our calculations indicated that the reduction of carbon dioxide could be carried out via two spin states, the high-spin (HS) triplet state and the low-spin (LS) singlet state. The minimum energy crossing points (MECPs) on the seam of two intersecting PESs (potential energy surfaces) were searched out. Some interesting phenomena, such as the open-loop phenomenon, and the O-rebound process, were demonstrated to be the important causes of the spin crossover. All these calculations gave us insight into the essence of the related experiment from the macro point of view, and helped to verify which spin states the related complexes pertinent were in. All of these researches would help advance the development of efficient and structurally tailorable CO2 hydrogenation catalysts.

A sensitive LC-ESI-MS method for the determination of indapamide in human plasma: method and clinical applications.

A sensitive LC-ESI-MS method for the determination of indapamide in human plasma using glibenclamide as the internal standard (IS) was established. Following acidification with 1 M hydrochloric acid solution, plasma samples were extracted with ethyl acetate and separated on a C(18) column with a mobile phase of 10 mM ammonium acetate-methanol (22:78, v/v). Indapamide was determined using electrospray ionization in a single quadrupole mass spectrometer. LC-ESI-MS was performed in the selected ion monitoring (SIM) mode using target ions at m/z 364.3 for indapamide and m/z 492.4 for the IS. Calibration curves were linear over the ranges of 0.1-100 ng/ml for indapamide. The lower limit of quantification was 0.1 ng/ml. The intra- and inter-assay precisions were less than 9.5% and 10.6%, respectively. The mean plasma extraction recovery of indapamide was 90.5-93.9%. The method has been successfully applied to study the pharmacokinetics of indapamide in healthy male Chinese volunteers.