Pharmacological characterization of the small molecule 03A10 as an inhibitor of α-synuclein aggregation for Parkinson's disease treatment.

Aggregation of α-synuclein, a component of Lewy bodies (LBs) or Lewy neurites in Parkinson's disease (PD), is strongly linked with disease development, making it an attractive therapeutic target. Inhibiting aggregation can slow or prevent the neurodegenerative process. However, the bottleneck towards achieving this goal is the lack of such inhibitors. In the current study, we established a high-throughput screening platform to identify candidate compounds for preventing the aggregation of α-synuclein among the natural products in our in-house compound library. We found that a small molecule, 03A10, i.e., (+)-desdimethylpinoresinol, which is present in the fruits of Vernicia fordii (Euphorbiaceae), modulated aggregated α-synuclein, but not monomeric α-synuclein, to prevent further elongation of α-synuclein fibrils. In α-synuclein-overexpressing cell lines, 03A10 (10 µM) efficiently prevented α-synuclein aggregation and markedly ameliorated the cellular toxicity of α-synuclein fibril seeds. In the MPTP/probenecid (MPTP/p) mouse model, oral administration of 03A10 (0.3 mg· kg-1 ·d-1, 1 mg ·kg-1 ·d-1, for 35 days) significantly alleviated behavioral deficits, tyrosine hydroxylase (TH) neuron degeneration and p-α-synuclein aggregation in the substantia nigra (SN). As the Braak hypothesis postulates that the prevailing site of early PD pathology is the gastrointestinal tract, we inoculated α-synuclein preformed fibrils (PFFs) into the mouse colon. We demonstrated that α-synuclein PFF inoculation promoted α-synuclein pathology and neuroinflammation in the gut and brain; oral administration of 03A10 (5 mg· kg-1 ·d-1, for 4 months) significantly attenuated olfactory deficits, α-synuclein accumulation and neuroinflammation in the olfactory bulb and SN. We conclude that 03A10 might be a promising drug candidate for the treatment of PD. 03A10 might be a novel drug candidate for PD treatment, as it inhibits α-synuclein aggregation by modulating aggregated α-synuclein rather than monomeric α-synuclein to prevent further elongation of α-synuclein fibrils and prevent α-synuclein toxicity in vitro, in an MPTP/p mouse model, and PFF-inoculated mice.

Distal mutation V486M disrupts the catalytic activity of DPP4 by affecting the flap of the propeller domain.

Dipeptidyl peptidase-4 (DPP4) plays a crucial role in regulating the bioactivity of glucagon-like peptide-1 (GLP-1) that enhances insulin secretion and pancreatic ß-cell proliferation, making it a therapeutic target for type 2 diabetes. Although the crystal structure of DPP4 has been determined, its structure-function mechanism is largely unknown. Here, we examined the biochemical properties of sporadic human DPP4 mutations distal from its catalytic site, among which V486M ablates DPP4 dimerization and causes loss of enzymatic activity. Unbiased molecular dynamics simulations revealed that the distal V486M mutation induces a local conformational collapse in a ß-propeller loop (residues 234-260, defined as the flap) and disrupts the dimerization of DPP4. The "open/closed" conformational transitions of the flap whereby capping the active site, are involved in the enzymatic activity of DPP4. Further site-directed mutagenesis guided by theoretical predictions verified the importance of the conformational dynamics of the flap for the enzymatic activity of DPP4. Therefore, the current studies that combined theoretical modeling and experimental identification, provide important insights into the biological function of DPP4 and allow for the evaluation of directed DPP4 genetic mutations before initiating clinical applications and drug development.

Characteristics of Chinese Families in Which Children and Both Parents Are Diagnosed with Malignant Tumors: A Retrospective Study.

Objective To characterize Chinese families in which both parents and at least one child are diagnosed with malignant diseases and provide reference for cancer screening or early detection in people whose both parents are diagnosed with cancer. Methods Medical records of all clients to the center of cancer screening and prevention of the National Cancer Center/Cancer Hospital between January 2008 and February 2018 were screened to select families in which both parents and at least one child were diagnosed with malignant diseases. The cancer profiles of fathers, mothers, sons and daughters, their age distribution at diagnosis, and similarity of cancers between two generations were analyzed. The proportions of each cancer in males and females of the cohort were compared with corresponding data from the National Cancer Center Registry of China (NCCRC) in 2013. Results Totally 135 families were identified from records of 33 200 clients. Proportion of lung cancer in fathers (40/135, 29.6%) and in mothers (38/135, 28.1%) were higher than the national data (23.9% in males and 14.9% in females, respectively). The proportion of breast cancer in daughters (35/109, 32.1%) was higher than that of mothers (14/135, 10.4%) and the national data (17.1%). In 71 father-son pairs of cancer, 46.5% (33/71) were of the same systematic disease, and 16.9% (12/71) were of the same cancer. These two indexes were 31.2% (n=34) and 10.1% (n=11), respectively in the 109 father-daughter pairs of cancer, 36.6% (n=26) and 8.5% (n=6) respectively in the 71 mother-son pairs of cancer, and 31.2% (n=34) and 20.2% (n=20) respectively in the 109 mother-daughter pairs of cancer. Sons were more likely to suffer from cancers originated from the same system as father's cancer than daughters (χ 2=4.299, P<0.05), and daughters were more likely to suffer from the same cancer as their mother's cancer than sons (χ 2=4.506, P<0.05). The age (mean ± standard deviation) of the daughters (52.4±12.7) and the sons (59.4±10.9) at diagnosis were significantly younger than the fathers (65.5±12.2) and the mothers (65.7 ±12.5) (all P<0.001). Conclusions For people whose both parents are diagnosed as cancer, screening or early detection examinations should cover a full range of cancers rather than the cancers their father and mother have suffered, or cancers stemmed from the same system as their parent's cancers. We suggest screening or early detection program for these special population start earlier than that for the general population, with emphasis on cancers derived from digestive system for males and women-specific cancers, i.e., breast cancer, ovarian cancer, cervical cancer and uterine cancer for females.

Docking-based structural splicing and reassembly strategy to develop novel deazapurine derivatives as potent B-RafV600E inhibitors.

The mutation of B-RafV600E is widespread in a variety of human cancers. Its inhibitors vemurafenib and dabrafenib have been launched as drugs for treating unresectable melanoma, demonstrating that B-RafV600E is an ideal drug target. This study focused on developing novel B-RafV600E inhibitors as drug leads against various cancers with B-RafV600E mutation. Using molecular modeling approaches, 200 blockbuster drugs were spliced to generate 283 fragments followed by molecular docking to identify potent fragments. Molecular structures of potential inhibitors of B-RafV600E were then obtained by fragment reassembly followed by docking to predict the bioactivity of the reassembled molecules. The structures with high predicted bioactivity were synthesized, followed by in vitro study to identify potent B-RafV600E inhibitors. A highly potent fragment binding to the hinge area of B-RafV600E was identified via a docking-based structural splicing approach. Using the fragment, 14 novel structures were designed by structural reassembly, two of which were predicted to be as strong as marketed B-RafV600E inhibitors. Biological evaluation revealed that compound 1m is a potent B-RafV600E inhibitor with an IC50 value of 0.05 µmol/L, which was lower than that of vemurafenib (0.13 µmol/L). Moreover, the selectivity of 1m against B-RafWT was enhanced compared with vemurafenib. In addition, 1m exhibits desirable solubility, bioavailability and metabolic stability in in vitro assays. Thus, a highly potent and selective B-RafV600E inhibitor was designed via a docking-based structural splicing and reassembly strategy and was validated by medicinal synthesis and biological evaluation.

[Rapid Propagation in vitro of Dioscorea opposita 'Guangfeng'and Its Stomatal Observation,FCM Analysis of Chromosome Ploidy and ISSR Detection of DNA Mutation].

Objective: To study the rapid propagation in vitro of Dioscorea opposita'Guangfeng', and to observe the stomas of the transplanting plantlets and potted seedlings, to test chromosome ploidy by FCM, and to detect DNA mutation by ISSR,in order to provide the technical basis for the large-scale production of Dioscorea opposita 'Guangfeng' plantlets. Methods: The technique system of Dioscorea opposita 'Guangfeng'rapid propagation in vitro was established and optimized by plant tissue culture method. The parameters of transplanting plantlets and potted seedlings were studied as follows, the stomatal parameters were observed by transparent adhesive tape method, chromosome ploidy were analyzed by FCM, and DNA mutation were detected by ISSR molecular marker. Results: The technique system of Dioscorea opposite 'Guangfeng' rapid propagation in vitro was as follows, slightly woody stem segment with a bud were selected and inoculated onto MS + KT 1 mg / L + NAA 0. 2 mg / L solid culture medium and cultured in the photoperiod of 14 h / d( the temperature was( 25 ± 2) ℃ and light intensity was 1 500 ~ 2 000 Lx) after disinfected for 1 min in 70% alcohol prior to sterilized for 12 min with 0. 1% Hg Cl2,the materials were washed with sterile water for 3 times, respectively. The new bud was cut off when it grew to 2 ~ 3cm and inoculated into MS + KT 2 mg / L + NAA 0. 5 mg / L liquid culture medium and continued to culture in above culture conditions. The whole plant was formed after cultured for about 90 d. The sealing membrane was opened in transplanting, and the plantlets was still placed in above culture conditions and cultured for 2 ~ 3 d, and then the whole plant was taken out, and the culture medium washed off and then transferred into the vessel with shallow liquid MS basic culture medium and domesticated indoor. The acclimated plantlets were taken out and transplanted in the outdoor pots with the sandy soil when the new shoots grew out, and watered one time with tap water in the morning and evening per day, the survival rate reached 100%. The results of stomatal observation, FCM analysis and ISSR detection of transplanting plantlets and potted seedlings showed that the stomatal parameters, chromosome ploidy and DNA mutation of plantlets and potted seedlings had no variation. Conclusion: The results reveal that the establishment and optimization of the technique system of Dioscorea opposita 'Guangfeng' rapid propagation in vitro is feasible, and the regenerated plants do not have genetic variation which can ensure the stability of the genetic.

[Effect of PEG-6000 Simulation Drought Stress on Physiological Characteristics of Guangfeng Medicinal Yam Plantlets].

Objective: In order to provide the theoretical basis for the Guangfeng medicinal yam( Dioscorea opposita) in field transplanting, the effect of PEG-6000 simulation drought stress on physiological characteristics of Guangfeng medicinal yam plantlets was studied. Methods: Using the method of spectrophotometer,the content of total chlorophyll,soluble total sugar, soluble protein and praline,as well as the activities of SOD,CAT and POD of Guangfeng medicinal yam plantlets were tested under PEG-6000 treatment. Results: Under PEG-6000 simulated drought stress, with the increasing of drought stress and the extension of stress time, the total chlorophyll content of Guangfeng medicinal yam plantlets continued to decline, the content of total soluble sugar, proline and MDA of Guangfeng medicinal yam plantlets significantly increased, the content of soluble protein and the activities of CAT,POD and SOD of Guangfeng medicinal yam plantlets increased at first and then decreased. Conclusion: This study reveals the changes of physiological indices of Guangfeng medicinal yam plantlets under PEG-6000 simulation drought stress, which indicated that Guangfeng medicinal yam plantlets have certain drought tolerance.

Inhibitory Effects of (2'R)-2',3'-dihydro-2'-(1-hydroxy-1-methylethyl)-2,6'-bibenzofuran-6,4'-diol on Mushroom Tyrosinase and Melanogenesis in B16-F10 Melanoma Cells.

(2'R)-2',3'-Dihydro-2'-(1-hydroxy-1-methylethyl)-2,6'-bibenzofuran-6,4'-diol (DHMB) is a natural compound extracted from Morus notabilis. It was found that DHMB acts as a competitive inhibitor against mushroom tyrosinase with a Ki value of 14.77 µM. Docking results further indicated that it could form strong interactions with one copper ion with a distance of 2.7 Å, suggesting the mechanism of inhibition might be due to chelating copper ions in the active site. Furthermore, melanin production in B16-F10 murine melanoma cells was significantly inhibited by DHMB in a concentration-dependent manner without cytotoxicity. The results of western blotting also showed that DHMB decreased 3-isobuty-1-methxlzanthine-induced mature tyrosinase expression. Taken together, these findings indicated that DHMB may be a new promising pigmentation-altering agent for agriculture, cosmetic, and therapeutic applications.

[Study on Rapid Micropropagation in Vitro Technique of Guangfeng Medicinal Yam (Dioscorea opposita) Plantlets].

OBJECTIVE: In order to provide methodology reference for virus-free and germplasm conservation of Guangfeng medicinal yam (Dioscorea opposita) plantlets, rapid micropropagation in vitro technique of Guangfeng medicinal yam plantlets was studied. METHODS: Using the method of plant tissue culture, single factor test and flow-cytometry, the basic procedure of Guangfeng medicinal yam tissue culture was established and the DNA content of Guangfeng medicinal yam plantlets and its potted seedlings was detected. RESULTS: The best disinfection procedure of stems with a bud of Guangfeng medicinal yam was washed with sterile water for three times after sterilized with 70% alcohol for 20 - 30 s and then washed with sterile water for three times again after sterilized with 0.1% mercuric chloride for 10 - 12 min; The best explants of stems with a bud of Guangfeng medicinal yam was slightly woody and more mature stems witha bud; The best proliferation culture medium of stems with a bud of Guangfeng medicinal yam was MS + 6-BA 2.0 mg/L + NAA 0.1 mg/L; The best rooting culture medium of stems with a bud of Guangfeng medicinal yam was MS + NAA 0.5 mg/L; The best culture method of Guangfeng medicinal yam plantlets was liquid culture; The best transplanting matrix of Guangfeng medicinal yam plantlets was the mixture of paddy clay and fine sand (1: 2) or the mixture of perlite and vermiculite (1: 2); The DNA content between Guangfeng medicinal yam plantlets and its potted seedlings had no significant difference. CONCLUSION: A fast and efficient micropropagation in vitro technological system of stems with a bud of Guangfeng medicinal yam is established, and the flow cytometry detect results also show the genetic stability of Guangfeng medicinal yam plantlets, whose results provide the technical and theoretical basis for the large-scale production of Guangfeng medicinal yam plantlets.

Multi-algorithm and multi-model based drug target prediction and web server.

AIM: To develop a reliable computational approach for predicting potential drug targets based merely on protein sequence. METHODS: With drug target and non-target datasets prepared and 3 classification algorithms (Support Vector Machine, Neural Network and Decision Tree), a multi-algorithm and multi-model based strategy was employed for constructing models to predict potential drug targets. RESULTS: Twenty one prediction models for each of the 3 algorithms were successfully developed. Our evaluation results showed that ∼30% of human proteins were potential drug targets, and ∼40% of putative targets for the drugs undergoing phase II clinical trials were probably non-targets. A public web server named D3TPredictor (http://www.d3pharma.com/d3tpredictor) was constructed to provide easy access. CONCLUSION: Reliable and robust drug target prediction based on protein sequences is achieved using the multi-algorithm and multi-model strategy.

Two new sulfated sesquiterpenoids from Petasites tricholobus.

Two new sulfated sesquiterpenoids, megastigman-7-ene-3, 5, 6, 9-tetrol-3-O-ß-D-6'-sulfonated-glucopyranoside (1) and 3-O-ß-D-6'-sulfonated-glucopyranosyl-6-(3-oxo-2-butenylidenyl)-1, 1, 5-trimethylcyclohexan-5-ol (2), along with one known sesquitepenoid compound icariside B1 (3) were isolated from the whole herb of Petasites tricholobus Franch. Their structures were identified by their chemical and spectroscopic characters. All obtained compounds were tested for their cytotoxicity against four cancer cell lines.