Investigating the Inhibition of Diindolylmethane Derivatives on SARS-CoV-2 Main Protease.

The SARS-CoV-2 main protease (Mpro) is an essential enzyme that promotes viral transcription and replication. Mpro conserved nature in different variants and its nonoverlapping nature with human proteases make it an attractive target for therapeutic intervention against SARS-CoV-2. In this work, the interaction mechanism between Mpro and diindolylmethane derivatives was investigated by molecular docking, enzymatic inhibition assay, UV-vis, fluorescence spectroscopy, and circular dichroism spectroscopy. Results of IC50 values show that 1p (9.87 µM) was the strongest inhibitor for Mpro in this work, which significantly inhibited the activity of Mpro. The binding constant (4.07 × 105 Lmol-1), the quenching constant (5.41 × 105 Lmol-1), and thermodynamic parameters indicated that the quenching mode of 1p was static quenching, and the main driving forces between 1p and Mpro are hydrogen bond and van der Waals force. The influence of molecular structure on the binding is investigated. Chlorine atoms and methoxy groups are favorable for the diindolylmethane derivative inhibitors of Mpro. This work confirms the changes in the microenvironment of Mpro by 1p, and provides clues for the design of potential inhibitors.

Size-dependent reactivity of VnO+ (n = 1-9) clusters with ethane.

Cost-effective and readily accessible 3d transition metals (TMs) have been considered as promising candidates for alkane activation while 3d TMs especially the early TMs are usually not very reactive with light alkanes. In this study, the reactivity of Vn+ and VnO+ (n = 1-9) cluster cations towards ethane under thermal collision conditions has been investigated using mass spectrometry and density functional theory calculations. Among Vn+ (n = 1-9) clusters, only V3-5+ can react with C2H6 to generate dehydrogenation products and the reaction rate constants are below 10-13 cm3 molecule-1 s-1. In contrast, the reaction rate constants for all VnO+ (n = 1-9) with C2H6 significantly increase by about 2-4 orders of magnitude. Theoretical analysis evidences that the addition of ligand O affects the charge distribution of the metal centers, resulting in a significant increase in the cluster reactivity. The analysis of frontier orbitals indicates that the agostic interaction determines the size-dependent reactivity of VnO+ cluster cations. This study provides a novel approach for improving the reactivity of early 3d TMs.

Investigation of steric hindrance effect on the interactions between four alkaloids and HSA by isothermal titration calorimetry and molecular docking.

The binding of four alkaloids with human serum albumin (HSA) was investigated by isothermal titration calorimetry (ITC), spectroscopy and molecular docking techniques. The findings demonstrated that theophylline or caffeine can bind to HAS, respectively. The number of binding sites and binding constants are obtained. The binding mode is a static quenching process. The effects of steric hindrance, temperature, salt concentration and buffer solution on the binding indicated that theophylline and HSA have higher binding affinity than caffeine. The fluorescence and ITC results showed that the interaction between HSA and theophylline or caffeine is an entropy-driven spontaneous exothermic process. The hydrophobic force was the primary driving factor. The experimental results were consistent with the molecular docking data. Based on the molecular structures of the four alkaloids, steric hindrance might be a major factor in the binding between HSA and these four alkaloids. This study elucidates the mechanism of interactions between four alkaloids and HSA.

Cytokine expression in gingival crevicular fluid around teeth opposing dental implants and 3-unit fixed partial dentures in a cross-sectional study.

OBJECTIVE: This study aimed to study the cytokines in gingival crevicular fluid (GCF) of the teeth opposing to dental implants and 3-unit fixed partial dentures (FPDs). MATERIALS AND METHODS: A total of 74 participants were recruited for this cross-sectional study. Based on the status of lower first molars, the participants were divided into dental implants group and 3-unit FPDs group. Social index and oral hygiene were recorded. Occlusal loading was evaluated with a T-scan. GCF was sampled from the upper first molar and assessed with a commercial cytokine assay kit. RESULTS: Forty three dental implants patients and 31 3-unit FPDs patients received all of the clinical and laboratory evaluation. The dental implants group had a higher occlusion force distribution on first molars region. IL-10, IL-17, RANK had a higher mean in dental implants group and was associated with occlusion force of first molar. There was a weakly association between IL-10 and dental implants in the binary logistic regression analyses. CONCLUSIONS: In this study, the teeth opposing implants have a higher level of cytokines in the GCF than teeth opposing to 3-unit FPDs in periodontal healthy participants because of the poor osseoperception of dental implants. IL-10 might reflect a higher occlusion force in dental implants region. CLINICAL RELEVANCE: This study provided that different tooth restoration methods could influence the periodontal status of the contact teeth.

Comparative study of the binding between chlorogenic acid and four proteins by isothermal titration calorimetry, spectroscopy and docking methods.

As a polyphenolic compound, chlorogenic acid has antioxidant, anti-inflammatory, antiviral, anti-obesity and other effects. Based on the interactions between chlorogenic acid and the proteins (human serum albumin (HSA), pepsin (Pep), trypsin (Try), fat mass and obesity-associated protein (FTO)), results will provide clues for screening effective inhibitors. The interaction between chlorogenic acid and the four proteins (HSA, Pep, Try, FTO) was analyzed by the aid of fluorescence quenching, synchronous fluorescence, three-dimensional fluorescence, isothermal titration calorimetry, and molecular docking. It can be concluded that there is no obvious interaction between chlorogenic acid and FTO. The binding affinity between chlorogenic acid and three proteins is HSA > Try > Pep. The binding process is spontaneous, and the quenching type is static quenching. Hydrophobic interaction and hydrogen bonding is observed in the binding process. This study provides valuable information for understanding the interaction mechanism between chlorogenic acid and proteins, and provides clues for screening inhibitors.

Structural characteristics of small-molecule inhibitors targeting FTO demethylase.

Studies have shown that the FTO gene is closely related to obesity and weight gain in humans. FTO is an N6-methyladenosine demethylase and is linked to an increased risk of obesity and a variety of diseases, such as acute myeloid leukemia, type 2 diabetes, breast cancer, glioblastoma and cervical squamous cell carcinoma. In light of the significant role of FTO, the development of small-molecule inhibitors targeting the FTO protein provides not only a powerful tool for grasping the active site of FTO but also a theoretical basis for the design and synthesis of drugs targeting the FTO protein. This review focuses on the structural characteristics of FTO inhibitors and discusses the occurrence of obesity and cancer caused by FTO gene overexpression.

The role of chlorine atom on the binding between acrylonitrile derivatives and fat mass and obesity-associated protein.

In this work, seven acrylonitrile derivatives were selected as potential inhibitors of fat and obesity-related proteins (FTO) by the aid of fluorescence spectroscopy, ultraviolet visible spectroscopy, molecular docking, and cytotoxicity methods. Results show that the interaction between 3-amino-2-(4-chlorophenyl)-3-phenylacrylonitrile (1a) and FTO was the strongest among these derivatives. Thermodynamic analysis and molecular modeling show that the main force between 1a and FTO is hydrophobic interaction. The cytotoxicity test showed that the IC50 value of 1a was 46.64 µmol/L, which indicated 1a had the smallest IC50 value and had the best inhibitory effect on the proliferation of leukemia K562 cells among the seven derivatives. Both our previous results and this work show that chlorine atoms play important role in the binding of small molecules and FTO. This work brings new information for the study of FTO inhibitors.

A Filter Supported Surface-Enhanced Raman Scattering "Nose" for Point-of-Care Monitoring of Gaseous Metabolites of Bacteria.

This work designs a convenient method for fabrication of surface-enhanced Raman scattering (SERS) devices by loading gold nanostars (AuNSs) on a flat filter support with vacuum filtration. The dense accumulation of AuNSs results in a strong sensitization to SERS signal and shows sensitive response to gaseous metabolites of bacteria, which produces a SERS "nose" for rapid point-of-care monitoring of these metabolites. The "nose" shows good reproducibility and stability and can be used for SERS quantitation of a gaseous target with Raman signal. The impressive performance of the proposed SERS "nose" for detecting gaseous metabolites of common foodborne bacteria like Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa from inoculated samples demonstrates its much higher sensitivity than that of human sense and application in distinguishing spoiled food at an early stage and real-time tracing of food spoilage degree. The strong point-of-care testing ability of the designed SERS "nose" and the miniaturization of whole equipment extend greatly the analytical application of SERS technology in food safety and public health.

"Three-in-One" SERS Adhesive Tape for Rapid Sampling, Release, and Detection of Wound Infectious Pathogens.

The traditional colony culture method for detection of pathogens is subjected to the laborious and tedious experimental procedure, which limits its application in point-of-care (POC) testing and quick diagnosis. This work designs an intelligent adhesive tape as a "three-in-one" platform for rapid sampling, photocontrolled release, and surface-enhanced Raman scattering (SERS) detection of pathogens from infected wounds. This tape is constructed by encapsulating densely packed gold nanostars as SERS substrates between two pieces of graphene and modified with a synthetic o-nitrobenzyl derivative molecule to form an artificial biointerface for highly efficient pathogen capture via electrostatic interaction. The captured targets can be conveniently released onto a solid culture medium by UV cleavage of o-nitrobenzyl moiety for pathogen growth and in situ SERS detection. As a proof of strategy, this "three-in-one" platform has been used for detecting the concurrent infection of Pseudomonas aeruginosa and Staphylococcus aureus by pasting the tape on a skin burn wound. The impressive detection performance with an analytical time of only several hours for these pathogens at an early growth stage demonstrates its great potential as a POC testing device for health care.

Identification of Clausine E as an inhibitor of fat mass and obesity-associated protein (FTO) demethylase activity.

The alkaloids containing a carbazole nucleus are an established class of natural products with wide range of biological activities. A combination of thermodynamic and enzymatic activity studies provides an insight into the recognition of Clausine E by the fat mass and obesity-associated protein (FTO). The binding of Clausine E to FTO was driven by positive entropy and negative enthalpy changes. Results also indicated that the hydroxyl group was crucial for the binding of small molecules with FTO. The structural and thermodynamic information provides the basis for the design of more effective inhibitors for FTO demethylase activity.

Cardioprotection of (±)-sodium 5-bromo-2-(α-hydroxypentyl) benzoate (BZP) on mouse myocardium I/R injury through inhibiting 12/15-LOX-2 activity.

(±)-Sodium5-bromo-2-(α-hydroxypentyl) benzoate (brand name: brozopine, BZP, 1a), derived from L-3-n-butylphthalide (L-NBP), has been reported to protect the brain from stoke and has been approved by CFDA in Phase I-II clinical trials. However, it remains to be investigated whether 1a may exhibit any cardioprotective effect on ischemia-reperfusion (I/R) injury. In the current study, C57BL/6 and ICR mice were pretreated with 1a, and myocardium I/R were then performed. We found that 1a not only significantly reduced the infarct size and improved cardiac contractile function after acute MI/R in both species, but also protected hearts from chronic MI-related cardiac injury. Mechanically, we found that 1a physically binds to 12/15-LOX-2 using molecular docking. The shRNA-mediated 12/15-LOX-2 knockdown almost completely blocked the protective effect of 1a. Our findings, for the first time, strongly indicate that 1a may serve as a potent and promising cardioprotective agent in treatment of I/R related injury, at least partially through targeting 12/15-LOX-2.

Mediation of Extracellular Polymeric Substances in Microbial Reduction of Hematite by Shewanella oneidensis MR-1.

Extracellular electron transfer (EET) plays a fundamental role in microbial reduction/oxidation of minerals. Extracellular polymeric substances (EPS) surrounding the cells constitute a matrix that separates the cell's outer membrane from insoluble minerals and environmental fluid. This study investigated the effects of EPS on EET processes during microbial reduction of hematite by the iron-reducing strain Shewanella oneidensis MR-1 (MR-1). Electrochemical characterization techniques were employed to determine the influence of EPS components on the redox ability of MR-1. Cells with removed EPS exhibited approximately 30% higher hematite reduction than regular MR-1 cells, and produced a current density of 56 µA cm-2, corresponding to 3-4 fold that of regular MR-1. The superior EET of EPS-deprived cells could be attributed to direct contact between outer membrane proteins and hematite surface, as indicated by more redox peaks being detected by cyclic voltammetry and differential pulse voltammetry. The significantly reduced current density of MR-1 cells treated with proteinase K and deoxyribonuclease suggests that the electron transfer capacity across the EPS layer depends mainly on the spatial distribution of specific proteins and electron shuttles. Exopolysaccharides in EPS tend to inhibit electron transfer, however they also favor the attachment of cells onto hematite surfaces. Consistently, the charge transfer resistance of cells lacking EPS was only 116.3 Ω, approximately 44 times lower than that of regular cells (5,139.1 Ω). These findings point to a negative influence of EPS on EET processes for microbial reduction/oxidation of minerals.

From aggregation-induced to solution emission: a new strategy for designing ratiometric fluorescent probes and its application for in vivo HClO detection.

In this paper, we introduced a new strategy for converting aggregation-induced emission (AIE) to fluorescence emission in solution into the rational design of new fluorescent probes. Two fluorescent probes based on this strategy, namely, PDAM-Lyso and PDAM-Me, have been synthesized and tested both in vitro and in vivo. The fluorophores of the two probes are both phenothiazine molecules, which link to the diaminomaleonitrile (DAMN) moiety through imine bonds. In the presence of imine bonds, the probes emit red fluorescence in an aqueous solution caused by the AIE effect. As the imine bonds are selectively cut-off by HClO, the DAMN moiety gets removed, inducing blue fluorescence of the reaction product. In this way, the selectivity of the DAMN-based probes toward HClO against metal ions and other reactive oxygen species (ROS) was successfully improved. The imaging of endogenous and exogenous HClO with these two probes reveals that lysosome-targeting probes are of great advantage in the detection of natively generated HClO. Furthermore, the imaging of endogenous HClO in zebrafish suggests that PDAM-Lyso is capable of monitoring the generation of HClO in vivo, illustrating that this strategy is of great significance in designing new probes.

Identification of nafamostat mesilate as an inhibitor of the fat mass and obesity-associated protein (FTO) demethylase activity.

Nafamostat mesilate is a serine protease inhibitor and used for the treatment of pancreatitis and cancers. The fat mass and obesity-associated protein (FTO) was identified to be a demethylase and played an important role in physiological processes. The aim of this study was to examine the inhibition of Nafamostat mesilate on FTO demethylase activity. A combination of thermodynamic and enzymatic activity studies provides an insight into the recognition of Nafamostat mesilate by FTO. The binding of Nafamostat mesilate to FTO was driven by higher positive entropy changes and smaller negative enthalpy changes. The structural and thermodynamic information provides the basis for the design of more effective inhibitors for FTO. Our results might provide a novel target protein for Nafamostat mesilate.

Comparative study of the bindings between 3-phenyl-1H-indazole and five proteins by isothermal titration calorimetry, spectroscopy and docking methods.

In this study, the interaction between 3-phenyl-1H-indazole (1a) and the fat mass and obesity-associated (FTO) protein was confirmed by isothermal titration calorimetry (ITC). The structure feature of 1a was different from our previously reported FTO inhibitors (radicicol, N-CDPCB and CHTB); the Cl and diol group in structure motif is critical for inhibitors to bind to FTO. In order to test whether there is specificity for the interaction between FTO and 1a, the interactions between 1a and four important proteins (human serum albumin (HSA), pepsin, catalase and trypsin) were investigated by ITC, spectroscopy and molecular docking methods. ITC results showed spontaneous exothermic reactions occurring between 1a and the proteins except trypsin under investigated conditions. The order of the binding affinity of 3-phenyl-1H-indazole is catalase > HSA > FTO > pepsin. Comparison between ITC and spectral results was made. This work will provide the basis for the design of novel inhibitors for FTO. Abbreviations CAT catalase DMSO dimethyl sulfoxide FTO fat mass and obesity-associated protein HSA human serum albumin Pep pepsin Try trypsin Communicated by Ramaswamy H. Sarma.

The role of chlorine atom on the binding between 2-phenyl-1H-benzimidazole analogues and fat mass and obesity-associated protein.

In this work, nine 2-phenyl-1H-benzimidazole structural analogues were screened for potential inhibitor of the fat mass and obesity-associated protein (FTO) by isothermal titration calorimetry (ITC). The results show that the binding between 6-chloro-2-phenyl-1H-benzimidazole (1d) and FTO was dominated by entropy. Results of enzymatic activity assays provided an IC50 value of 24.65 µM for 1d. Our previous results and comparison of nine structural analogues indicated that the chlorine atom was crucial for the binding of small molecules with FTO. The identification of novel small molecules may provide information for the design of FTO inhibitors and the treatment of leukemia.

Identification of Natural Compound Radicicol as a Potent FTO Inhibitor.

The fat mass and obesity-associated protein (FTO), as an m6A demethylase, is involved in many human diseases. Virtual screening and similarity search in combination with bioactivity assay lead to the identification of the natural compound radicicol as a potent FTO inhibitor, which exhibits a dose-dependent inhibition of FTO demethylation activity with an IC50 value of 16.04 µM. Further ITC experiments show that the binding between radicicol and FTO was mainly entropy-driven. Crystal structure analysis reveals that radicicol adopts an L-shaped conformation in the FTO binding site and occupies the same position as N-CDPCB, a previously identified small molecular inhibitor of FTO. Unexpectedly, however, the 1,3-diol group conserved in radicicol and N-CDPCB assumes strikingly different orientations for interaction with FTO. The identification of radicicol as an FTO inhibitor and revelation of its recognition mechanism not only opens the possibility of developing new therapeutic strategies for treatment of leukemia but also provide clues for elucidation of the acting mechanisms of radicicol, which is a possible clinical candidate worth in-depth study.

bifA Regulates Biofilm Development of Pseudomonas putida MnB1 as a Primary Response to H2O2 and Mn2.

Pseudomonas putida (P. putida) MnB1 is a widely used model strain in environment science and technology for determining microbial manganese oxidation. Numerous studies have demonstrated that the growth and metabolism of P. putida MnB1 are influenced by various environmental factors. In this study, we investigated the effects of hydrogen peroxide (H2O2) and manganese (Mn2+) on proliferation, Mn2+ acquisition, anti-oxidative system, and biofilm formation of P. putida MnB1. The related orthologs of 4 genes, mco, mntABC, sod, and bifA, were amplified from P. putida GB1 and their involvement were assayed, respectively. We found that P. putida MnB1 degraded H2O2, and quickly recovered for proliferation, but its intracellular oxidative stress state was maintained, with rapid biofilm formation after H2O2 depletion. The data from mco, mntABC, sod and bifA expression levels by qRT-PCR, elucidated a sensitivity toward bifA-mediated biofilm formation, in contrary to intracellular anti-oxidative system under H2O2 exposure. Meanwhile, Mn2+ ion supply inhibited biofilm formation of P. putida MnB1. The expression pattern of these genes showed that Mn2+ ion supply likely functioned to modulate biofilm formation rather than only acting as nutrient substrate for P. putida MnB1. Furthermore, blockade of BifA activity by GTP increased the formation and development of biofilms during H2O2 exposure, while converse response to Mn2+ ion supply was evident. These distinct cellular responses to H2O2 and Mn2+ provide insights on the common mechanism by which environmental microorganisms may be protected from exogenous factors. We postulate that BifA-mediated biofilm formation but not intracellular anti-oxidative system may be a primary protective strategy adopted by P. putida MnB1. These findings will highlight the understanding of microbial adaptation mechanisms to distinct environmental stresses.

A novel porphyrin-based near-infrared fluorescent probe for hypochlorite detection and its application in vitro and in vivo.

Reactive oxygen species (ROS), especially HOCl/ClO-, have been demonstrated to play essential roles in both physiological and pathological processes, and an abnormal level of HOCl/ClO- is related to some diseases. In this work, a very fast responsive (within 30 seconds) porphyrin-based fluorescent probe, TPP-TCF, for ClO- with a NIR emissive wavelength was prepared. This probe exhibited excellent selectivity towards ClO- and would not be interfered with by other ROS and typical nucleophiles. The limit of detection (LOD) for ClO- was evaluated to be 0.29 µM, indicating high sensitivity towards ClO-. In further bioimaging experiments, TPP-TCF displayed low-cytotoxicity and good cell penetrability for recognizing exogenous ClO- in HeLa cells. Moreover, this probe was successfully applied in imaging endogenous ClO- in living animals.

A small molecular pH-dependent fluorescent probe for cancer cell imaging in living cell.

A novel pH-dependent two-photon fluorescent molecular probe ABMP has been prepared based on the fluorophore of 2, 4, 6-trisubstituted pyridine. The probe has an absorption wavelength at 354 nm and corresponding emission wavelength at 475 nm with the working pH range from 2.20 to 7.00, especially owning a good liner response from pH = 2.40 to pH = 4.00. ABMP also has excellent reversibility, photostability and selectivity which promotes its ability in analytical application. The probe can be excited with a two-photon fluorescence microscopy and the fluorescence cell imaging indicated that the probe can distinguish Hela cancer cells out of normal cells with a two-photon fluorescence microscopy which suggested its potential application in tumor cell detection.