[Advancements in virtual screening techniques for study of enzyme inhibitor compounds].

Enzymes are closely associated with the onset and progression of numerous diseases, making enzymes a primary target in innovative drug development. However, the challenge remains in identifying compounds that exhibit potent inhibitory effects on the target enzymes. With the continuous expansion of the total number of natural products and increasing difficulty in isolating and enriching new compounds, traditional high-throughput screening methods are finding it increasingly challenging to meet the demands of new drug development. Virtual screening, characterized by its high efficiency and low cost, has gradually become an indispensable technology in drug development. It represents a prominent example of the integration of artificial intelligence with biopharmaceuticals and is an inevitable trend in the rapid development of innovative drug screening in the future. Therefore, this article primarily focused on systematically reviewing the recent applications of virtual screening technology in the development of enzyme inhibitors and explored the prospects and advantages of using this technology in developing new drugs, aiming to provide essential theoretical insights and references for the application of related technologies in the field of new drug development.

Synthesis, molecular docking, and evaluation of antibacterial activity of 1,2,4-triazole-norfloxacin hybrids.

A series of 1,2,4-triazole-norfloxacin hybrids was designed, synthesized, and evaluated for in vitro antibacterial activity against common pathogens. All the newly synthesized compounds were characterized by Fourier-transform infrared spectrophotometry, proton and carbon nuclear magnetic resonance, and electrospray ionization-mass spectrometry. Representative compounds from each step of the synthesis were further characterized by X-ray crystallography. Many of the compounds synthesized exhibited antibacterial activity superior to that of norfloxacin toward both, gram-positive and gram-negative bacteria. The toxicity of the 1,2,4-triazole-norfloxacin hybrids toward bacterial cells was 32-512 times higher than that toward mouse fibroblast cells. Moreover, hemolysis was not observed at concentrations of 64 µg/mL, suggesting good biocompatibility. Molecular docking showed a least binding energy of -9.4 to -9.7 kcal/mol, and all compounds were predicted to show remarkable affinity for the bacterial topoisomerase IV.

Preparation of S-iron-enriched yeast using siderophores and its effect on iron deficiency anemia in rats.

Efforts to obtain organic trace elements have been made, including yeast enrichment and transformation, but the application of yeast for this purpose is restricted by poor tolerance and low enrichment. Siderophores play an important role in iron transport. Thus, the role of siderophores in iron transport under high-iron conditions and the application of siderophores in the enrichment of elements was explored. The results showed that some siderophores from iron-tolerant strains promoted yeast growth and increased its intracellular iron content. Among them, siderophore TZT-12 (from LK1110) was the best for promoting yeast growth and iron conversion. The siderophore-iron-enriched yeast (S-iron-enriched yeast) effectively restored the iron concentration, and an iron concentration of 59.40 mg/g was obtained by adding TZT-12. Iron deficiency anemia in rats was significantly mitigated with S-iron-enriched yeast compared with ferrous sulfate. These findings provide a new perspective on the preparation of organic trace elements for supplementation or food fortification.

Application of enzyme-assisted extraction of baicalin from Scutellaria baicalensis Georgi.

Endophytes may depend on degrading the plant cell wall with cellulases for their survival. Therefore, cellulase produced by endophytes may be useful in releasing the active ingredient of medicinal plants. Scutellaria baicalensis Georgi is a traditional Chinese medicinal plant widely used in China and baicalin is one of its main active ingredients. In this study, fresh S. baicalensis Georgi was used to isolate endophytes, Congo red staining was used to screen cellulase-producing strains, and HPLC was used to determine the content of baicalin in S. baicalensis Georgi. As a result, a highly active strain of endophyte capable of the extraction of high levels of baicalin was obtained. The strain was named HG-5 and identified as Bacillus sp. Scanning electron microscopy analysis confirmed that the enzyme better promotes the dissolution of plant active ingredients. After optimizing the enzyme production and extraction processes, we found that when compared with the traditional extraction method, the baicalin yield was increased 79.31% after extraction with the HG-5 enzyme. The current study provides a novel approach and method for the use of endophyte cellulase to improve the extraction of compounds from medicinal plants.

Diversity and Bioactivity of Endophytes From Angelica sinensis in China.

Plant seeds are not merely reproductive organs, they are also carriers of microorganism, particularly, inherent and non-invasive characteristic endophytes in host plant. Therefore, in this study, the endophytic diversity of Angelica seeds was studied and compared with endophytes isolated from healthy leaves, stems, roots, and seeds of A. sinensis using 20 different media. The metabolites of endophytic strains were evaluated with six different methods for their antioxidant activity and the paper disc diffusion method for antimicrobial activities. As a result, 226 endophytes were isolated. Compared with the biodiversity and abundance of uncultured fungi from Angelica seed, the result showed that the most frequent endophytic fungi were Alternaria sp. as seen in artificial media; moreover, compared with artificial media, the pathogenic fungi, including Fusarium sp. and Pseudallescheria sp., were not found from the Angelica seed, the results suggested it may not be inherent endophytes in plants. In addition, bacteria from seven phyla were identified by high-throughput sequencing, while five phyla of endophytic bacteria were not isolated on artificial media including Proteobacteria, Actinobacteria, Bacteroidetes, Microgenomates, and Saccharibacteria. Furthermore, the sample JH-4 mycelium displayed the best antioxidant activity, and the active constituent may be a flavonoid as determined by total phenol and flavonoid content. Moreover, YH-12-1 mycelium had strong inhibitory activity against the five tested strains and the minimum inhibitory concentration (MIC) against Pseudomonas aeruginosa and Streptococcus pneumoniae was found to be 25 µg/mL. Our results confirm that plant endophytes are rich in biodiversity and contain important resource of many uncultured microorganisms.

A new mixed inhibitor of adenosine deaminase produced by endophytic Cochliobolus sp. from medicinal plant seeds.

Medicinal plants have been studied for potential endophytic interactions and numerous studies have provided evidence that seeds harbor diverse microbial communities, not only on their surfaces but also within the embryo. Adenosine deaminase (ADA) is known as a potential therapeutic target for the treatment of lymphoproliferative disorders and cancer. Therefore, in this study, 20 types of medicinal plant seeds were used to screen endophytic fungi with tissue homogenate and streak. In addition, 128 morphologically distinct endophyte strains were isolated and their ADA inhibitory activity determined by a spectrophotometric assay. The strain with the highest inhibitory activity was identified as Cochliobolus sp. Seven compounds were isolated from the strain using a chromatography method. Compound 3 showed the highest ADA inhibitory activity and was identified as 5-hydroxy-2-hydroxymethyl-4H-pyran-4-one, based on the results of 1H and 13C NMR spectroscopy. The results of molecular docking suggested that compound 3 binds to the active site and the nonspecific binding site of the ADA. Furthermore, we found that compound 3 is a mixed ADA inhibitor. These results indicate that endophytic strains are a promising source of ADA inhibitors and that compound 3 may be a superior source for use in the preparation of biologically active ADA inhibitor compounds used to treat cancer.

Reynoutria Japonica from Traditional Chinese Medicine: A Source of Competitive Adenosine Deaminase Inhibitors for Anticancer.

BACKGROUND: Adenosine deaminase (ADA) is an important enzyme in purine metabolism and is known as a potential therapeutic target for the treatment of lymphoproliferative disorders and cancer. Traditional Chinese Herbal Medicine (TCHM) is widely used alone or in combination with chemotherapy to treat cancer, due to its ability to deliver a broad variety of bioactive secondary metabolites as promising sources of novel organic natural agents. OBJECTIVE: In the present study, 29 varieties of medicinal plants were screened for the presence of ADA inhibitors. RESULTS: Extracts from Reynoutria japonica, Glycyrrhiza uralensis, Lithospermum erythrorhizon, Magnolia officinalis, Gardenia jasminoides, Stephania tetrandra, Commiphora myrrha, Raphanus sativus and Corydalis yanhusuo demonstrated strong ADA inhibition with rates greater than 50%. However, Reynoutria japonica possessed the highest ADA inhibitory activity at 95.26% and so was used in our study for isolating the ADA inhibitor to be further studied. Eight compounds were obtained and their structures were identified. The compound H1 had strong ADA inhibitory activity and was deduced to be emodin by 1H and 13C-NMR spectroscopic analysis with an IC50 of 0.629 mM. The molecular docking data showed that emodin could bind tightly to the active site of ADA. Our results demonstrated that emodin displayed a new biological activity which is ADA inhibitory activity with high cytotoxic activity against K562 leukemia cells. The bioactivity of cordycepin was significantly increased when used in combination with emodin. CONCLUSION: Emodin may represent a good candidate anti-cancer therapy and adenosine protective agent.

Colletotrichine B, a new sesquiterpenoid from Colletotrichum gloeosporioides GT-7, a fungal endophyte of Uncaria rhynchophylla.

One new compound, colletotrichine B (1), was produced by the fungal Colletotrichum gloeosporioides GT-7. The structure of 1 was elucidated on the basis of spectroscopic analysis and X-ray crystallographic analysis. Monoamine oxidase (MAO), acetylcholinesterase (AChE) and phosphoinositide 3-kinase (PI3Kα) inhibitory activity of 1 was also evaluated. Compound 1 showed only AChE inhibiting activity with IC50 value of 38.0 ± 2.67 µg/mL.

Identification of a New Uncompetitive Inhibitor of Adenosine Deaminase from Endophyte Aspergillus niger sp.

Adenosine deaminase (ADA) is an enzyme widely distributed from bacteria to humans. ADA is known as a potential therapeutic target for the treatment of lymphoproliferative disorders and cancer. Endophytes are endosymbionts, often bacteria or fungi, which live within plant tissues and internal organs or intercellular space. Endophytes have a broad variety of bioactive metabolites that are used for the identification of novel natural compounds. Here, 54 morphologically distinct endophyte strains were isolated from six plants such as Peganum harmala Linn., Rheum officinale Baill., Gentiana macrophylla Pall., Radix stephaniae tetrandrae, Myrrha, and Equisetum hyemale Linn. The isolated strains were used for the search of ADA inhibitors that resulted in the identification of the strain with the highest inhibition activity, Aspergillus niger sp. Four compounds were isolated from this strain using three-step chromatography procedure, and compound 2 was determined as the compound with the highest inhibition activity of ADA. Based on the results of 1H and 13C NMR spectroscopies, compound 2 was identified as 3-(4-nitrophenyl)-5-phenyl isoxazole. We showed that compound 2 was a new uncompetitive inhibitor of ADA with high cytotoxic effect on HepG2 and SMCC-7721 cells (the IC50 values were 0.347 and 0.380 mM, respectively). These results suggest that endophyte strains serve as promising sources for the identification of ADA inhibitors, and compound 2 could be an effective drug in the cancer treatment.

A novel enzyme-assisted approach for efficient extraction of Z-ligustilide from Angelica sinensis plants.

Endophytes coexist with plants, in part, due to cellulase that allow saccharification of plant cell walls. The cellulase enzymes found in naturally occurring endophytes may exhibit stronger activity and more specificity than commercially available cellulase for enzyme-assisted extraction of compounds from medicinal plant materials. In order to identify endophytes with high cellulase activity, we screened endophytes taken from different parts of Angelica sinensis using the Congo red staining method. We identified three strains with higher cellulase activity. Of the 3 strains identified, No.Lut1201 increased the yield of extracted Z-ligustilide 2 fold compared to commercially available cellulase (Ningxia Sunson) using a cellulase-assisted extraction method and traditional extraction methods. Scanning electron microscopy clearly demonstrated that the cellulase extracted from endophytes enhance cell wall polysaccharide degradation as well as Z-ligustilide extraction from Radix Angelica sinensis (RAS). The current study provides a new method and ideas of using cellulase of endophytes for improving the extraction of compounds from medicinal plants.

Antitumor activity of a Rhodococcus sp. Lut0910 isolated from polluted soil.

The actinomycetes strain, lut0910, was isolated from polluted soil and identified as the Rhodococcus species with 99% similarity based on the sequence analysis of 16S recombinant DNA. The extract of this strain demonstrated in vivo and in vitro antitumor activity. The treatment of two human cancer cell lines, hepatocellular carcinoma HepG2 and cervical carcinoma Hela cells, with the lut0910 extract caused the delay in cell propagation in a dose-dependent manner with an IC50 of 73.39 and 33.09 µg/mL, respectively. Also, the oral administration of lut0910 extract to the mice with a solid tumor resulted in the inhibition of tumor growth in comparison with a placebo group. The thymus and spleen indexes were significantly increased in mice groups treated with the lut0910 extract. The histopathological changes of the tumor tissues showed that there were massive necrotic areas in the tumor tissues after treatment with different doses of the lut0910 extract. Our result would provide a new way and potent source for development of new anticancer agent from the polluted environment.

Expression and Purification of Delta Sleep-Inducing Peptide Fused with Protein Transduction Domain and Human Serum Albumin in Pichia pastoris.

BACKGROUND: Sleep is a natural part of every individual's life. Delta sleep-inducing peptide (DSIP) is a nonapeptide that could promote sleep through the induction of slow wave sleep. However, little is known about the pharmacological effect of DSIP on insomnia. OBJECTIVES: The main objective of this study was to analyze the pharmacological effect of DSIP on insomnia. METHODS: We designed a fusion protein containing N-terminal TAT-based transduction domain followed by human serum albumin and DSIP and designated this protein as PHD fusion protein. The PHD fusion protein were expressed in Pichia pastoris and purified. Mice were administered single subcutaneous injections three concentrations of PHD fusion protein (0.5, 1, 2 mg/kg), and the pharmacological activity of PHD fusion protein was studied using classic pentobarbitalinduced sleep test. RESULTS: We expressed the PHD fusion protein in P. pastoris; furthermore, the PHD fused protein was purified to near homogeneity by DEAE Sepharose FF, Phenyl Sepharose HP and Blue Sepharose 6 FF. Our result showed that the increase of pentobarbital-induced hypnotic effect characterized by reducing sleep latency and prolonged sleep duration was observed for increasing concentrations of PHD fusion protein (P<0.05); moreover, different dose of PHD fusion protein could induce the mice to re-sleep in a dose-dependent manner, whereas higher doses of PHD fusion protein (1.0, 2.0 mg/kg) significantly increased the rate of sleep re-onset compared with the vehicle group of mice (P<0.05). CONCLUSION: PHD fusion protein increased the hypnotic effects of pentobarbital by reducing sleep latency and prolonged sleep duration. The present study suggested PHD fusion protein could be a new drug candidate for insomnia.

Design and Hardware Implementation of a New Chaotic Secure Communication Technique.

In this paper, a scheme for chaotic modulation secure communication is proposed based on chaotic synchronization of an improved Lorenz system. For the first time, the intensity limit and stability of the transmitted signal, the characteristics of broadband and the requirements for accuracy of electronic components are presented by Multisim simulation. In addition, some improvements are made on the measurement method and the proposed experimental circuit in order to facilitate the experiments of chaotic synchronization, chaotic non-synchronization, experiment without signal and experiment with signal. To illustrate the effectiveness of the proposed scheme, some numerical simulations are presented. Then, the proposed chaotic secure communication circuit is implemented through analog electronic circuit, which is characterized by its high accuracy and good robustness.

Effects of Fe-YM1504 on iron deficiency anemia in rats.

Iron deficiency anemia (IDA) is one of the most serious forms of malnutrition. It is possible that some strains present in the natural environment possess a higher tolerance to inorganic iron and a higher ability to convert and accumulate iron compared with Saccharomyces cerevisiae wild-type strain. In the present study, the strain no. YM1504, able to grow in an iron-rich environment, was used as a potential organic iron supplement, and its efficacy in alleviating IDA in rats was investigated. Sixty female weanling Sprague-Dawley rats were randomly divided into a normal control group fed with a standard diet and a model group fed with an iron-deficient diet to create the IDA model. After the model was established, IDA rats were further randomly divided into five subgroups: the IDA group, the ferrous sulfate (FeSO4) group and Fe-YM1504 low-, medium- or high-dose groups receiving different concentrations of Fe-YM1504 supplements. Our results showed that Fe-YM1504 has an effective restorative function by returning the hemoglobin (Hb), hematocrit (HCT), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), serum iron (SI), total iron binding capacity (TIBC), serum ferritin (SF), etc. in IDA animals to the normal level. Moreover, malondialdehyde and the enzyme activities of superoxide dismutase and glutathione peroxidase in both plasma and liver homogenate were improved. Finally, compared with the FeSO4 group, the Fe-YM1504 middle-dose was more effective in alleviating IDA and fewer side effects were observed. The present study indicated that iron-enriched strain no. YM1504 might play a significant role in ameliorating IDA rats and might be exploited as a new iron supplement.

Higher Bioavailability of Organic Bound Zinc from High Zinc-Enriched Fungi.

The organic forms of trace elements are considered more bioavailable than the inorganic forms. Although yeast can enrich metal elements and convert inorganic zinc to organic species, its tolerability and transforming capacity are limited. It would therefore be very interesting to look for higher conversion and accumulation in zinc fungi to obtain organic bound zinc from the natural environment. In this paper, potato dextrose agar (PDA) medium containing 800 µg/mL zinc was used for initial screening, with twenty-two fungal strains that tolerated high zinc isolated from the natural environment, and one strain (No.LZ-1108) growing well at a zinc (II) concentration of 10,000 µg/mL. According to morphological analysis, 18S rDNA sequence analysis, and biophysical and biochemical characteristics, No.LZ-1108 was tentatively identified as Fusarium oxysporum. Using atomic absorption spectrometry, the zinc content in the No.LZ-1108 cells was found to be 6.7 mg/g dry cell. After oral administration to rats at a dose of 10 mg Zn (II)/kg body weight, the area under the plasma concentration-time curve (AUC) and the maximum zinc blood concentration (Cmax) of No.LZ-1108 and zinc gluconate were 8.10 g/L.min and 4.28 g/L.min, 23.72 µg/mL and 6.23 µg/mL, respectively. The AUC of No.LZ-1108 was significantly higher than those of zinc gluconate (P<0.05), and the mean relative bioavailability of AUC(test)/AUC(zinc gluconate) was 190 %, which showed that the bound zinc in No.LZ-1108 was more bioavailable than zinc gluconate. The present study reports an interesting alternative to developing zinc-based supplements from a natural source of zinc.

Separation and isolation of fusion protein using a new native preparative PAGE device.

A human serum albumin and Thymosin α1 (HSA-Tα1) fusion protein was designed and over-expressed in Pichia pastoris. To purify the fusion protein, a new native preparative electrophoresis system that involved a modified device with a sample receiving chamber, and an assay method with Coomassie Blue G-250 tracing the collection of the protein of interest. In this device, two gels were run in parallel: native vertical collecting polyacrylamide gel electrophoresis (PAGE) and native vertical tracing PAGE. Samples mixed with or without Coomassie Blue G-250 loading buffer were separately loaded to the two aforementioned gels, and the fractions were collected until the tracing protein band combined with dye reached 1 cm from the sample-receiving chamber at the bottom of the gel. Approximately nine fractions were collected at regular intervals of 15 min. HSA-Tα1 fusion protein with 95% relative homogeneity was harvested and manifested similar immunological activities as synthetic Tα1 after a single-step purification of this preparative PAGE. As a result, this system offers a new, rapid and simple method for the purification of the protein of interest.

Bioactivity and pharmacokinetics of two human serum albumin-thymosin alpha1-fusion proteins, rHSA-Talpha1 and rHSA-L-Talpha1, expressed in recombinant Pichia pastoris.

Thymosin-alpha1 (Talpha1) is indicated for the treatment of certain viral infections, including hepatitis B and C, and cancers, such as melanoma. In this paper, the fusion genes encoding human serum albumin (HSA) and Talpha1 with (rHSA-L-Talpha1) and without a linker peptide (rHSA-Talpha1) were constructed and overexpressed in P. pastoris. Through the process of ion interaction chromatography (Q-Sepharose F.F), hydrophobic interaction chromatography (Phenyl Sepharose HP) and affinity chromatography (Blue Sepharose F.F), the purity of fusion proteins was greater than 97%. In contrast to the reactivity of normal spleen cells to Con A, the data of in vitro murine spleen lymphocytes proliferation experiment suggested that spleen cells achieved a higher degree of T cell maturation after rHSA-L-Talpha1, rHSA-Talpha1 and Talpha1 treatments, respectively. Moreover, rHSA-L-Talpha1, rHSA-Talpha1 and Talpha1 can also antagonize dexamethasone-induced apoptosis of thymocyte sub-populations. In hydrocortisone-induced immunosuppression mice (in vivo experiments), after subcutaneous injections with two fusion proteins and Talpha1 for seven consecutive days, the net increment of body weight, the spleen index and the thymus index were significantly improved. Simultaneously, the increase in SOD level and the decrease in MDA level in plasma were observed. The pharmacokinetic data of rHSA-L-Talpha1 and rHSA-Talpha1 administered in rats showed an improved pharmacokinetic profile with a conspicuous prolonged half life. The analysis of bioactivity and pharmacokinetics suggested that fusion proteins rHSA-L-Talpha1 and rHSA-Talpha1 were new drug candidates.

[Establishment of a new gastric bypass animal-model with GK rats].

OBJECTIVE: To establish a new gastric bypass animal-model with Goto-Kakizaki rats whose different parts of the small intestine were bypassed while stomach was not bypassed. METHODS: Forty male 3-month-old GK rats were randomly divided into 4 groups: group I (sham operation), group II (duodenum bypassed), group III (jejunum bypassed), group IV (ileum bypassed). Fasting plasma glucose was measured before operation and the 1st, 4th,and 8th week after operation in all the rats, the body weight of all the rats were measured simultaneously. RESULTS: The survival rate of operation for the rats was 95%. Two rats in group IV (died on the first day after operation. The mean fasting plasma glucose concentration of the rats in group II, III, IV (declined obviously 4 weeks after gastric bypass [group II (12.02+/-1.97) vs (6.36+/-0.50) mmol/L, group III (13.42+/-1.66) vs (5.96+/-0.53) mmol/L, group IV (14.32+/-2.82) vs (5.18+/-0.49) mmol/L, all P <0.01], but there were no significant differences among the gastric bypassed groups. The weight of rats in group I, II, III (increased obviously after gastric bypass [group I (253.6+/-9.37) vs (367.0+/-23.70) g, group II (268.2+/-7.95) vs (384.8+/-16.12) g, group III (253.0+/-6.20) vs (323.0+/-16.40) g, all P <0.05] except the rats in group IV ([(262.0+/-13.47) vs(185.8+/-11.56) g]. CONCLUSIONS: The mean fasting plasma glucose concentration of the GK rats decreases obviously after gastric bypass through different parts of small intestine. The fasting plasma glucose concentration is not associated with the length of small intestine and body weight.

[Association between toll like receptor 4 (896A>G) mutations and pancreatic necrotic infection in severe acute pancreatitis].

OBJECTIVE: To determine the relationship between Toll like receptor (TLR) 4 (896A>G) mutations and pancreatic necrotic infection in acute pancreatitis (AP). METHODS: TLR4 (896A>G) mutations were detected by mispairing PCR-RFLP analysis technique in the patients with pancreatic necrosis and healthy volunteers. RESULTS: (1) All of the TLR4 mutations were heterozygotes; (2) The G allele frequencies of TLR4 genes were significantly higher in the patients with pancreatic infection than in the healthy volunteers; (3) The incidence of gram-negative infection was significantly higher in the patients with the TLR4 mutations [15 (44.1%) of 34] than that in the wild type population [15(18.5%) of 81]. CONCLUSION: TLR4 (896A>G) mutations are associated with the infection of pancreatic necrosis in the patients with AP.