Functional metalloenzymes based on myoglobin and neuroglobin that exploit covalent interactions.

Globins, such as myoglobin (Mb) and neuroglobin (Ngb), are ideal protein scaffolds for the design of functional metalloenzymes. To date, numerous approaches have been developed for enzyme design. This review presents a summary of the progress made in the design of functional metalloenzymes based on Mb and Ngb, with a focus on the exploitation of covalent interactions, including coordination bonds and covalent modifications. These include the construction of a metal-binding site, the incorporation of a non-native metal cofactor, the formation of Cys/Tyr-heme covalent links, and the design of disulfide bonds, as well as other Cys-covalent modifications. As exemplified by recent studies from our group and others, the designed metalloenzymes have potential applications in biocatalysis and bioconversions. Furthermore, we discuss the current trends in the design of functional metalloenzymes and highlight the importance of covalent interactions in the design of functional metalloenzymes.

Design, synthesis and evaluation of C-5 substituted pyrrolopyridine derivatives as potent Janus Kinase 1 inhibitors with excellent selectivity.

The development of highly selective Janus Kinase 1 (JAK1) inhibitors is crucial for improving efficacy and minimizing adverse effects in the clinical treatment of autoimmune diseases. In a prior study, we designed a series of C-5 4-pyrazol substituted pyrrolopyridine derivatives that demonstrated significant potency against JAK1, with a 10 âˆ¼ 20-fold selectivity over Janus Kinase 2 (JAK2). Building on this foundation, we adopted orthogonal strategy by modifying the C-5 position with 3-pyrazol/4-pyrazol/3-pyrrol groups and tail with substituted benzyl groups on the pyrrolopyridine head to enhance both potency and selectivity. In this endeavor, we have identified several compounds that exhibit excellent potency and selectivity for JAK1. Notably, compounds 12b and 12e, which combined 4-pyrazol group at C-5 site and meta-substituted benzyl tails, displayed IC50 value with 2.4/2.2 nM and high 352-/253-fold selectivity for JAK1 over JAK2 in enzyme assays. Additionally, both compounds showed good JAK1-selective in Ba/F3-TEL-JAK1/2 cell-based assays. These findings mark a substantial improvement, as these compounds are 10-fold more potent and over 10-fold more selective than the best compound identified in our previous study. The noteworthy potency and selectivity properties of compounds 12b and 12e suggest their potential utility in furthering the development of drugs for autoimmune diseases.

Regulating the Heme Active Site by Covalent Modifications: Two Case Studies of Myoglobin.

Using myoglobin (Mb) as a model protein, we herein developed a facial approach to modifying the heme active site. A cavity was first generated in the heme distal site by F46 C mutation, and the thiol group of Cys46 was then used for covalently linked to exogenous ligands, 1H-1,2,4-triazole-3-thiol and 1-(4-hydroxyphenyl)-1H-pyrrole-2,5-dione. The engineered proteins, termed F46C-triazole Mb and F46C-phenol Mb, respectively, were characterized by X-ray crystallography, spectroscopic and stopped-flow kinetic studies. The results showed that both the heme coordination state and the protein function such as H2 O2 activation and peroxidase activity could be efficiently regulated, which suggests that this approach might be generally applied to the design of functional heme proteins.

Based on the interaction between supramolecules of traditional Chinese medicine and enterobacteria to explore the material basis of combination of Rhei Radix et Rhizoma - Coptidis Rhizoma / 药学学报

Based on the interaction between supramolecule of traditional Chinese medicine and enterobacteria, the material basis of Rhei Radix et Rhizoma and Coptidis Rhizoma was explored. Scanning electron microscopy (SEM) and dynamic light scattering (DLS) were used to characterize the morphological differences of Rhubarb single decoction, Coptis single decoction and Rhubarb and Coptis co-decoction. An in vitro antibacterial model (E. coli, E. faecium and B. subtilis) was established to evaluate the damage effect of the combination of Rhei Radix et Rhizoma and Coptidis Rhizoma on enterobacteria. Ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to analyze the changes of chemical components of single decoctions and co-decoctions. The co-decoction of Rhei Radix et Rhizoma and Coptidis Rhizoma was turbid after decocting. The spherical particles of 300-400 nm were observed under SEM, and the co-decoction was more uniform and stable than that of single decoction. The interaction between supramolecules formed after the combination of Rhei Radix et Rhizoma and Coptidis Rhizoma and enterobacteria was significantly different from that of single decoction. In the process of interaction between supramolecules and enterobacteria, the spherical state was maintained, and the medicinal ingredients in Coptidis Rhizoma or Rhei Radix et Rhizoma were blocked, which could effectively alleviate the damage to enterobacteria. This study provided a reference for subsequent studies on the regulation of intestinal flora homeostasis by the combination of Rhei Radix et Rhizoma and Coptidis Rhizoma.

Peroxidase activity of a Cu-Fe bimetallic hydrogel and applications for colorimetric detection of ascorbic acid.

A Cu-Fe bimetallic hydrogel (2-QF-CuFe-G) was constructed through a simple method. The 2-QF-CuFe-G metallohydrogel possesses excellent peroxidase-like activity to catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. The catalytic mechanism was confirmed by the addition of •OH radical scavenger isopropyl alcohol (IPA), tert-butyl alcohol (TBA) and ˙OH trapping agent terephthalic acid (TA). Remarkably, the resultant blue ox-TMB system can be used to selectively and sensitively detect ascorbic acid (AA) with an LOD of 0.93 µM in the range of 4-36 µM through the colorimetric method. Moreover, the assay based on the 2-QF-CuFe-G metallohydrogel can be successfully applied to detect AA in fresh fruits.

Application of engineered myoglobins for biosynthesis of clofazimine by integration with chemical synthesis.

Significant efforts have been made in the design of artificial metalloenzymes. Myoglobin (Mb), an O2 carrier, has been engineered to exhibit different functions. Herein, we applied a series of engineered Mb mutants with peroxidase activity for biosynthesis of clofazimine (CFZ), a potential drug with a broad-spectrum antiviral activity, by integration with chemical synthesis. Two of those mutants, F43Y Mb and F43Y/T67R Mb, have been shown to efficiently catalyze the oxidative coupling of 2-N-(4-chlorophenyl) benzene-1,2-diamine (N-4-CPBDA) in the presence of H2O2, with 97% yields. The overall catalytic efficiency (kcat/Km) is 46-fold and 82-fold higher than that of WT Mb, respectively. By further combination of this reaction with chemical synthesis, the production of CFZ was accomplished with an isolated yield of 72%. These results showed that engineered Mbs containing the Tyr-heme cross-link (F43Y Mb and F43Y/T67R Mb) exhibit enhanced activity in the oxidative coupling reaction. This study also indicates that the combination of biocatalysis and chemical synthesis avoids the need for the separation of intermediate products, which offers a convenient approach for the total synthesis of the biological compound CFZ.

Synthesis of CF3-Containing 2-Imidazolines and Imidazoles via a Iodide-Promoted [3 + 2] Cyclization Reaction.

We report herein a general and effective system achieving cyclization of ß-trifluoromethyl enones with amidines in the presence of 1,3-diiodo-5,5-dimethylhydantoin (DIH), which affords a range of trifluoromethylated 2-imidazolines in synthetically useful yields with good diastereoselectivities (up to 95% yield, up to 98:2 dr) and good functional group tolerance. Furthermore, the one-pot synthesis of trifluoromethylated imidazoles via sequential cyclization and oxidation is demonstrated. More significantly, the reaction mechanism was verified by ESI-MS studies of possible intermediates, and a reasonable reaction mechanism was proposed.

Exploiting and Engineering Neuroglobin for Catalyzing Carbene N-H Insertions and the Formation of Quinoxalinones.

It is desired to design and construct more efficient enzymes with better performance to catalyze carbene N-H insertions for the synthesis of bioactive molecules. To this end, we exploited and designed a series of human neuroglobin (Ngb) mutants. As shown in this study, a double mutant, A15C/H64G Ngb, with an additional disulfide bond and a modified heme active site, exhibited yields up to >99% and total turnover numbers up to 33000 in catalyzing the carbene N-H insertions for aromatic amine derivatives, including those with a large size such as 1-aminopyrene. Moreover, for o-phenylenediamine derivatives, they underwent two cycles of N-H insertions, followed by cyclization to form quinoxalinones, as confirmed by the X-ray crystal structures. This study suggests that Ngb can be designed into a functional carbene transferase for efficiently catalyzing carbene N-H insertion reactions with a range of substrates. It also represents the first example of the formation of quinoxalinones catalyzed by an engineered heme enzyme.

Protective effect of thymoquinone on glycation of human myoglobin induced by d-ribose.

Nonenzymatic glycation and the subsequent accumulation of advanced glycation end-products (AGEs) in proteins are factors underlying long-term pathogenesis in diabetes. The study of protein glycation is crucial for elucidating their relationship with diabetes mellitus and related disorders. This study explores the interaction between d-ribose and human myoglobin (HMb), as well as the protective effect of thymoquinone (TQ) on glycation. A time-dependent in-vitro glycation study was performed to investigate the mechanism of d-ribose-induced structural interference of HMb in the absence and presence of TQ. Spectroscopic and proteomic analysis indicated that the presence of TQ significantly reduced the total amount of AGEs while maintaining structural characteristics of HMb. 14 glycated sites on HMb were further identified via liquid chromatography-tandem mass spectrometry (LC-MS/MS) after incubation with d-ribose for 12 h, predominantly interacting with lysine residues. TQ was found to disrupt this interaction, reducing the glycated sites from 14 to 12 sites and the percentage of glycated peptides from 26.50 % to 12.97 %. Additionally, there was a significant decrease in the degree of glycation at the same sites. In summary, our findings suggest that TQ has the potential to act as an anti-glycation agent and provide a comprehensive understanding underlying the inhibition mechanism of glycation.

White-light-emitting Ln-hydrogels with multi-stimuli responsiveness and applications in environmental sensing and anti-counterfeiting.

The construction of smart materials, especially white light emitting (WLE) hydrogels with multi-stimuli responsive properties, has received widespread attention from researchers. In this study, a WLE hydrogel was obtained by the in situ doping of Eu3+ and Tb3+ into a blue emission low molecular weight gelator (MPF). Remarkably, the prepared WLE hydrogel possessed excellent stimuli responsiveness to pH, temperature and chemicals, and could be used as a soft thermometer and a selective sensor for Cu2+. The correlated color temperature of the WLE hydrogel was calculated to be 5063 K, suggesting a potential application in cool white light. Moreover, a series of metallohydrogels with different colors were obtained by modulating the ratio of MPF, Eu3+ and Tb3+ or changing the excitation wavelength, which was an excellent candidate to construct soft materials of a full-color system. Additionally, the WLE hydrogel could be used for constructing anti-counterfeiting materials. Therefore, this study provides a new approach for preparing smart WLE hydrogels with multiple functions.

Functional copper complexes with benzofurans tridentate ligand: Synthesis, crystal structure, DNA binding and anticancer studies.

Metal complexes, particularly copper(II) complexes, are often used as anticancer drugs due to their ability to generate reactive oxygen species (ROS) in cells. Four copper(II) complexes have been designed based on ligands for triplet pyridine derivatives (complexes 1-4), and their structures have been determined using X-ray single crystal analysis. The interactions of these complexes with calf thymus DNA (CT-DNA) have been investigated using various techniques, including UV-vis absorption, viscosity measurements, and circular dichroism spectroscopy. The results indicate that complexes 1-4 strongly interact with DNA through partial intercalations. Further investigation using agarose gel electrophoresis shows that all four complexes can cleave pBR322 DNA in the presence of ascorbic acid as a reducing agent, and the DNA cleavage mechanism is through the generation of singlet oxygen (1O2). In vitro anticancer activities of these complexes have been evaluated using A549, MDA-MB-231, HeLa, and HepG2 cells. The calculated IC50 values indicate significant efficacy against cancer cells. Additionally, AO/EB staining assays reveal that these complexes induce cell apoptosis in HeLa cell line.

Effects of naturally occurring S47F/A mutations on the structure and function of human cytochrome c.

The sequence and structure of human cytochrome c (hCyt c) exhibit evolutionary conservations, with only a limited number of naturally occurring mutations in humans. Herein, we investigated the effects of the naturally occurring S47F/A mutations on the structure and function of hCyt c in the oxidized form. Although the naturally occurring S47F/A mutations did not largely alter the protein structure, the S47F and S47A variants exhibited a small fraction of high-spin species. Kinetic studies showed that the peroxidase activity of the variants was enhanced by ∼2.5-fold under neutral pH conditions, as well as for the rate in reaction with H2O2, when compared to those of wild-type hCyt c. In addition, we evaluated the interaction between hCyt c and human neuroglobin (hNgb) by isothermal titration calorimetry (ITC) studies, which revealed that the binding constant was reduced by ∼8-fold as result of the mutation of the hydrophilic Ser to the hydrophobic Phe/Ala. These findings provide valuable insights into the role of Ser47 in Ω-loop C in sustaining the structure and function of hCyt c.

Discovery of C-5 Pyrazole-Substituted Pyrrolopyridine Derivatives as Potent and Selective Inhibitors for Janus Kinase 1.

Developing selective inhibitors for Janus kinase 1 (JAK1) is a significant focus for improving the efficacy and alleviating the adverse effects in treating immune-inflammatory diseases. Herein, we report the discovery of a series of C-5 pyrazole-modified pyrrolopyrimidine derivatives as JAK1-selective inhibitors. The potential hydrogen bond between the pyrazole group and E966 in JAK1 is the key point that enhances JAK1 selectivity. These compounds exhibit 10- to 20-fold JAK1 selectivity over JAK2 in enzyme assays. Compound 12b also exhibits excellent JAK1 selectivity in Ba/F3-TEL-JAK cellular assays. Metabolism studies and the results of the hair growth model in mice indicate that compound 12b may be a viable lead compound for the development of highly JAK1-selective inhibitors for immune and inflammatory diseases.

Self-oxidation of cysteine to sulfinic acid in an engineered T67C myoglobin: structure and reactivity.

Myoglobin (Mb) was found to undergo self-oxidation when a cysteine residue was engineered at position 67 in the heme distal site. Both the X-ray crystal structure and mass spectrum confirmed the formation of a sulfinic acid (Cys-SO2H). Moreover, the self-oxidation could be controlled during protein purification to yield the unmodified form (T67C Mb). Importantly, both T67C Mb and T67C Mb (Cys-SO2H) were able to be labeled by chemicals, which provided useful platforms to generate artificial proteins.

A fluorescent probe based on a phenylalanine derivative is capable of sequential detection of Zn2+ and Cys/His.

A facile and dual fluorescent chemosensor (named 7-IDF) based on a phenylalanine derivative with an indole group was designed and synthesized. 7-IDF can selectively and sensitively detect Zn2+ via obvious fluorescence enhancement in an aqueous solution. Remarkably, the 7-IDF-Zn complex with blue luminescence has higher selectivity toward cysteine (Cys) and histidine (His) than for other amino acids. Intriguingly, 7-IDF can also be used as an excellent probe to detect Zn2+ in real water samples. Moreover, 7-IDF and 7-IDF-Zn possess excellent biocompatibility and cell permeability, and 7-IDF can consecutively detect Zn2+ and Cys/His in Hela cells through fluorescence imaging experiments. This study suggests that the phenylalanine-based chemosensor possesses great potential applications for the sequential detection of Zn2+ and Cys/His in biosystems.

The oxidative nuclease activity of human cytochrome c with mutations in Ω-loop C/D.

Natural and artificial nucleases have extensive applications in biotechnology and biomedicine. The exploration of protein with potential DNA cleavage activity also inspires the design of artificial nuclease and helps to understand the physiological process of DNA damage. In this study, we engineered four human cytochrome c (Cyt c) mutants (N52S, N52A, I81N, and I81D Cyt c), which showed enhanced DNA cleavage activity and degradation in comparison with WT Cyt c, especially under acidic conditions. The mechanism assays revealed that the superoxide (O2•-) plays an important role in the nuclease reaction. The kinetic assays showed that the peroxidase activity of the I81D Cyt c mutant enhanced up to 9-fold at pH 5. This study suggests that the mutations of Ile81 and Asn52 in Ω-loop C/D are critical for the nuclease activity of Cyt c, which may have physiological significance in DNA damage and potential applications in biomedicine.

Covalent immobilizing horseradish peroxidase on electrochemically-functionalized biochar for phenol removal.

Enzyme-based biocatalytic treatment has been known as an effective measure to biologically degrade organic pollutants. Advantageously, enzymes could be immobilized on solid supports, and such fact enables reuse/prolong the enzymatic capability. It could be of great importance to functionalize a support material for enhancing the immobilization efficiency/stability of enzymes. As such, this study laid great emphasis on covalent bonding to immobilize horseradish peroxidase (HRP) on a functionalized rice straw biochar with glutaraldehyde (GA) as a crosslinker. Biochar was pretreated by the electrochemical method and the acid treatment respectively to enrich the oxygen-containing functional groups. These led to the enhanced immobilizing ability of biochar. The HRP immobilized on the electrochemically-functionalized biochar (HRP-EBC) showed three times as much enzyme activity as the HRP directly adsorbed onto biochar. The HRP immobilized on the acid-functionalized biochar (HRP-ABC) showed activity similar to that of HRP-EBC. It was concluded that both the (acid/electrochemical) pretreatments are effective to enhance enzyme immobilization. Nevertheless, the electrochemical functionalized method of biochar is chemical oxidant-free, and one important lesson from a series of tests was that the pretreatment of biochar through the electrochemical method could be more environmentally benign. Moreover, employing HRP-EBC could be beneficial from a perspective of a real environmental practice considering its higher pH, thermal stability, and good reusability. 80% of phenol was degraded in 1 h in the presence of HRP-EBC when pH was 7.0 and a ratio of H2O2 to phenol was 1:1.5.

Phenoxazinone Synthase-like Activity of Rationally Designed Heme Enzymes Based on Myoglobin.

The design of functional metalloenzymes is attractive for the biosynthesis of biologically important compounds, such as phenoxazinones and phenazines catalyzed by native phenoxazinone synthase (PHS). To design functional heme enzymes, we used myoglobin (Mb) as a model protein and introduced an artificial CXXC motif into the heme distal pocket by F46C and L49C mutations, which forms a de novo disulfide bond, as confirmed by the X-ray crystal structure. We further introduced a catalytic Tyr43 into the heme distal pocket and found that the F43Y/F46C/L49C Mb triple mutant and the previously designed F43Y/F46S Mb exhibit PHS-like activity (80-98% yields in 5-15 min), with the catalytic efficiency exceeding those of natural metalloenzymes, including o-aminophenol oxidase, laccase, and dye-decolorizing peroxidase. Moreover, we showed that the oxidative coupling product of 1,6-disulfonic-2,7-diaminophenazine is a potential pH indicator, with the orange-magenta color change at pH 4-5 (pKa = 4.40). Therefore, this study indicates that functional heme enzymes can be rationally designed by structural modifications of Mb, exhibiting the functionality of the native PHS for green biosynthesis.

The effects of natural polysaccharide supramolecular hydrogel on bacteriostatic activity and biofilm clearance of berberine / 药学学报

Taking berberine (BBR) as an example, to study whether the supramolecular hydrogel formed by berberine and lotus root starch (Nelumbo nucifera Gaertn; LRS), a natural polysaccharide, affects the inhibition to Staphylococcus aureus and the ability of biofilm clearance. The chemical structure and rheological properties of BBR@LRS gel were characterized by infrared spectroscopy and rheometer. The in vitro release of supramolecular hydrogel was observed at pH = 1.2 and pH = 7.4. Broth dilution method and biofilm clearence experiment were used to observe the bacteriostasis and biofilm clearance respectively. Cytotoxicity test and in vitro hemolysis test were used to evaluate the biosafety preliminarily. The results showed that the LRS polysaccharide hydrogel could encapsulate BBR, and there was an interaction between them. The BBR@LRS gel had good rheological properties and biosafety, and played a role in solubility enhancement and slow release of BBR, which was stronger than BBR in inhibiting the growth of Staphylococcus aureus and clearing biofilm. This study provides reference for the effect of natural polysaccharide supramolecular hydrogels on biological functions of active components of traditional Chinese medicine.

Exposure level of neonicotinoid pesticides and their metabolites in pregnant women in the suburb of Shanghai / 中华预防医学杂志

In 2021, a total of 151 pregnant women were selected from the suburb of Shanghai. A questionnaire survey was conducted to obtain data about maternal age, gestational week, total annual household income, education level and passive smoking among pregnant women and one spot urine was collected. The concentrations of eight neonicotinoid pesticides and four metabolites in urine were measured by ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry. The differences in detection frequencies and concentrations of neonicotinoid pesticides and their metabolites among pregnant women with different characteristics were compared, and the influencing factors of the detection of neonicotinoid pesticides in urine were analyzed. The results showed that at least one neonicotinoid pesticide was detected in 93.4% (141 samples) of urine samples. The detection frequencies of N-desmethyl-acetamiprid, clothianidin, thiamethoxam, and N-desmethyl-clothianidin were high, about 78.1% (118 samples), 75.5% (114 samples), 68.9% (104 samples), and 44.4% (67 samples), respectively. The median concentration of the sum of all neonicotinoid pesticides was 2.66 μg/g. N-desmethyl-acetamiprid had the highest detection concentration with a median concentration of 1.04 μg/g. A lower urinary detection frequency of imidacloprid and its metabolites was seen in pregnant women aged 30-44 years [OR (95%CI): 0.23 (0.07-0.77)]. A higher detection frequency of clothianidin and its metabolites was seen in pregnant women with per capita annual household income≥100 000 yuan [OR (95%CI): 6.15 (1.56-24.28)]. There was widespread exposure to neonicotinoid pesticides and their metabolites in pregnant women from the suburb of Shanghai, which might pose potential health risks to pregnant women, and maternal age and household income were potential influencing factors of the exposure to neonicotinoid pesticides.