An Integrated In Silico Method to Discover Novel Rock1 Inhibitors: Multi- Complex-Based Pharmacophore, Molecular Dynamics Simulation and Hybrid Protocol Virtual Screening.

Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) is an important regulator of focal adhesion, actomyosin contraction and cell motility. In this manuscript, a combination of the multi-complex-based pharmacophore (MCBP), molecular dynamics simulation and a hybrid protocol of a virtual screening method, comprised of multipharmacophore- based virtual screening (PBVS) and ensemble docking-based virtual screening (DBVS) methods were used for retrieving novel ROCK1 inhibitors from the natural products database embedded in the ZINC database. Ten hit compounds were selected from the hit compounds, and five compounds were tested experimentally. Thus, these results may provide valuable information for further discovery of more novel ROCK1 inhibitors.

Casitas B-Lineage Lymphoma RING Domain Inhibitors Protect Mice against High-Fat Diet-Induced Obesity and Insulin Resistance.

The casitas b-lineage lymphoma (c-Cbl) is an important adaptor protein with an intrinsic E3 ubiquitin ligase activity that interacts with E2 proteins such as UbCH7. c-Cbl plays a vital role in regulating receptor tyrosine kinase signaling. c-Cbl involves in whole-body energy homeostasis, which makes it a potential target for the treatment of type 2 diabetes and obesity. In the present study, we have designed two parental peptides and 55 modified peptides based on the structure of UbCH7 loop L1 and L2. Thirteen of the modified peptides showed increased inhibitory activity in a fluorescence polarization-based assay. In the in vivo proof of study principle, mice treated with peptides 10, 34, 49 and 51 were protected against high-fat diet-induced obesity and insulin resistant. These inhibitors may potentially lead to new therapeutic alternatives for obesity and type 2 diabetes.

Integrated in silico and experimental methods revealed that Arctigenin inhibited angiogenesis and HCT116 cell migration and invasion through regulating the H1F4A and Wnt/ß-catenin pathway.

Arctigenin (ARG) has been previously reported to exert diverse biological activities including anti-proliferation, anti-inflammatory, and antiviral, etc. In the current study, the anti-metastasis and anti-angiogenesis activities of ARG were investigated. To further understand how ARG played these bioactivities, proteomic approaches were used to profile the proteome changes in response to ARG treatment using 2DE-MS/MS. Using these approaches, a total of 50 differentially expressed proteins were identified and clustered. Bioinformatics analysis suggested that multiple signalling pathways were involved. Moreover, ARG induced anti-metastatic and anti-angiogenesis activities were mainly accompanied by a deactivation of the Wnt/ß-catenin pathway in HCT116 cells.

Development of a fluorescence polarization based high-throughput assay to identify Casitas B-lineage lymphoma RING domain regulators.

The E3 ubiquitin protein ligase Casitas B-lineage Lymphoma (Cbl) proteins and their binding partners play an important role in regulating signal transduction pathways. It is important to utilize regulators to study the protein-protein interactions (PPIs) between these proteins. However, finding specific small-molecule regulators of PPIs remains a significant challenge due to the fact that the interfaces involved in PPIs are not well suited for effective small molecule binding. We report the development of a competitive, homogeneous, high-throughput fluorescence polarization (FP) assay to identify small molecule regulators of Cbl (RING) domain. The FP assay was used to measure binding affinities and inhibition constants of UbCH7 peptides and small molecule regulators of Cbl (RING) domains, respectively. In order to rule out promiscuous, aggregation-based inhibition, two assay conditions were developed and compared side by side. Under optimized conditions, we screened a 10,000 natural compound library in detergent-free and detergent-present (0.01% Triton X-100) systems. The results indicate that the detergent-present system is more suitable for high-throughput screens. Three potential compounds, methylprotodioscin, leonuride and catalpol, have been identified that bind to Cbl (RING) domain and interfere with the Cbl (RING)-UbCH7 protein-protein interaction.

Identification of adiponectin receptor agonist utilizing a fluorescence polarization based high throughput assay.

Adiponectin, the adipose-derived hormone, plays an important role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. It has been shown that up-regulation of adiponectin or adiponectin receptor has a number of therapeutic benefits. Given that it is hard to convert the full size adiponectin protein into a viable drug, adiponectin receptor agonists could be designed or identified using high-throughput screening. Here, we report on the development of a two-step screening process to identify adiponectin agonists. First step, we developed a high throughput screening assay based on fluorescence polarization to identify adiponectin ligands. The fluorescence polarization assay reported here could be adapted to screening against larger small molecular compound libraries. A natural product library containing 10,000 compounds was screened and 9 hits were selected for validation. These compounds have been taken for the second-step in vitro tests to confirm their agonistic activity. The most active adiponectin receptor 1 agonists are matairesinol, arctiin, (-)-arctigenin and gramine. The most active adiponectin receptor 2 agonists are parthenolide, taxifoliol, deoxyschizandrin, and syringin. These compounds may be useful drug candidates for hypoadiponectin related diseases.

Simultaneous determination of oleanolic acid and ursolic acid by RP-HPLC in the leaves of Eriobotrya japonica Lindl.

Oleanolic acid (OA) and ursolic acid (UA) are isomeric triterpenic acids and only one methyl's position is different between them. OA and UA always exist in the same plant, so it is difficult to separate them when determining contents by RP-HPLC. In this study, a very simple mobile phase for HPLC was developed to simultaneously determine UA and OA, and the factors affecting separation were also discussed. The mobile phase is methanol: water (95:5) with flow rate 0.4 mL/min. The retention time for OA and UA was 20.58 and 21.57 min, respectively, the resolution was 1.61. The average contents of OA and UA of three Loquat leaves sets were 1.4 mg/g and 5.6 mg/g, respectively. Regarding the HPLC, we found that changing mobile phase, adjusting the pH value or adding ion-pairing agent could not affect the separation between UA and OA greatly. While adjustment of the flow rate and column temperature could improve the resolution greatly.

The association of SULT1A1 codon 213 polymorphism and breast cancer susceptibility: meta-analysis from 16 studies involving 23,445 subjects.

Epidemiological studies on the association between SULT1A1 codon 213 polymorphism and breast cancer risk are inconclusive. In order to derive a more precise estimation of the association, a meta-analysis was conducted in this article. Sixteen studies including 9,881 cases and 13,564 controls were collected for SULT1A1 codon 213 polymorphism by searching the databases of Medline, PubMed, Embase, and ISI Web of Knowledge. The strength of association between SULT1A1 codon 213 polymorphism and breast cancer susceptibility was assessed by calculating crude ORs with 95% CIs. When all the 21 studies were pooled into the meta-analysis, there was no evidence for significant association between SULT1A1 codon 213 polymorphism and breast cancer susceptibility (for Arg/Arg versus Arg/His: OR = 0.999, 95% CI = 0.941-1.061; for Arg/Arg versus His/His: OR = 1.121, 95% CI = 1.013-1.242; for dominant model: OR = 1.128, 95% CI = 1.01-1.26; for recessive model: OR = 1.151, 95% CI = 0.950-1.394). In the subgroup analysis by the source of controls, significant increased risk was found for hospital-based studies (for Arg/Arg versus Arg/His: OR = 1.173, 95% CI = 1.000-1.376; for Arg/Arg versus His/His: OR = 1.600, 95% CI = 1.134-2.256; for dominant model: OR = 1.269, 95% CI = 1.134-2.256; for recessive model: OR = 1.664, 95% CI = 1.070-2.588). In summary, the meta-analysis suggests that SULT1A1 codon 213 polymorphism may be associated with the hospital-based studies. However, large number of samples and representative hospital-based studies with homogeneous breast cancer patients and well-matched controls are warranted to confirm this finding.

Experimental investigation of the immunoregulatory and anti-inflammatory effects of the traditional Chinese medicine "Li-Yan Zhi-Ke Granule" for relieving chronic pharyngitis in rats.

Chronic pharyngitis, a chronic inflammation of the pharyngeal mucous membrane and submucous lymphoid tissues, is often caused by unsatisfactory treatment of acute pharyngitis or repeated occurrences of upper respiratory tract infection and is related to a high-dust environment. Traditional herbal pharmacotherapy is well known for combining plant species to create complex phytochemical mixtures in the attempt to ameliorate pathophysiological processes. The aim of current study is to investigate the effect of immunoregulation and anti-inflammation with the traditional Chinese medicine (TCM) "Li-Yan Zhi-Ke Granule" in rats. Determination of serum hemolysin and the carbon particle clearance test were performed. The results demonstrate that administration of the TCM "Li-Yan Zhi-Ke Granule" may improve the effect of phagocytosis by mononuclear macrophages and immune function in rats, and may also increase the immunoregulatory and anti-inflammatory responses of rats with chronic pharyngitis. This traditional drug could relieve the symptoms of sore throat and cough in rats with chronic pharyngitis.

Synthesis and characterization of graft copolymer of chitosan and polyethylene glycol.

MPEG was modified with 1,1'-carbonyldiimidazole, then the activated MPEG reacted with primary amino groups of chitosan. Synthesize the graft copolymer of chitosan and polyethylene glycol in two steps. The structure of the copolymer was characterized by FT-IR and 1H-NMR. It agrees with the PEG content of classical stealth nanoparticles materials. The X-ray diffraction and DSC analysis proved that the crystallinity of the copolymer increased. It is a promising material for the stealth nanoparticles. It is a potential new carrier for the drug delivery systems of long-circulation and solid carcinoma.

Differential proteomics identification of HSP90 as potential serum biomarker in hepatocellular carcinoma by two-dimensional electrophoresis and mass spectrometry.

The aim of the current study is to identify the potential biomarkers involved in Hepatocellular carcinoma (HCC) carcinogenesis. A comparative proteomics approach was utilized to identify the differentially expressed proteins in the serum of 10 HCC patients and 10 controls. A total of 12 significantly altered proteins were identified by mass spectrometry. Of the 12 proteins identified, HSP90 was one of the most significantly altered proteins and its over-expression in the serum of 20 HCC patients was confirmed using ELISA analysis. The observations suggest that HSP90 might be a potential biomarker for early diagnosis, prognosis, and monitoring in the therapy of HCC. This work demonstrates that a comprehensive strategy of proteomic identification combined with further validation should be adopted in the field of cancer biomarker discovery.

(25R)-5a-Spiro-stane-3,12-dione.

The title compound, C(27)H(40)O(4), was obtained from the oxidation of (25R)-3b-hydr-oxy-5a-spiro-stan-12-one (Hecogenin) by Jone's reagent. The mol-ecule contains six alicyclic and heterocyclic rings, all trans-fused, among which four six-membered rings adopt similar chair conformations while two five-membered rings assume an envelope conformation.